U.S. patent application number 12/932304 was filed with the patent office on 2011-11-24 for methods and compositions for inhibition of bcl6 repression.
Invention is credited to Khaja Farid Ahmad, Jonathan D. Licht, Ari M. Melnick, Gilbert Prive.
Application Number | 20110286928 12/932304 |
Document ID | / |
Family ID | 34700096 |
Filed Date | 2011-11-24 |
United States Patent
Application |
20110286928 |
Kind Code |
A1 |
Melnick; Ari M. ; et
al. |
November 24, 2011 |
Methods and compositions for inhibition of BCL6 repression
Abstract
Provided are peptides or mimetics that block corepressor binding
to a BCL6 lateral groove. Also provided are methods or blocking
corepressor binding to the BCL6 lateral groove. Additionally,
methods of inhibiting BCL6 repression in a mammalian cell, and
methods of treating a mammal with cancer are provided.
Inventors: |
Melnick; Ari M.; (New York,
NY) ; Licht; Jonathan D.; (North Brook, IL) ;
Prive; Gilbert; (Toronto, CA) ; Ahmad; Khaja
Farid; (San Francisco, CA) |
Family ID: |
34700096 |
Appl. No.: |
12/932304 |
Filed: |
February 23, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10582662 |
May 24, 2007 |
7919578 |
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PCT/US2004/042418 |
Dec 16, 2004 |
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12932304 |
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60530102 |
Dec 16, 2003 |
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Current U.S.
Class: |
424/9.2 ;
435/7.1; 435/8; 436/86 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 49/0056 20130101; A61K 38/10 20130101; A61K 47/549 20170801;
A61K 47/645 20170801 |
Class at
Publication: |
424/9.2 ; 436/86;
435/8; 435/7.1 |
International
Class: |
A61K 49/00 20060101
A61K049/00; G01N 33/53 20060101 G01N033/53; C12Q 1/66 20060101
C12Q001/66; A61P 35/00 20060101 A61P035/00; G01N 33/68 20060101
G01N033/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] The U.S. Government has a paid-up license in this invention
and the right in limited circumstances to require the patent owner
to license others on reasonable terms as provided by the terms of
CA 59936 AM and R21 CA 99982, both awarded by the National
Institutes of Health.
Claims
1-61. (canceled)
62. A method of determining whether a test compound inhibits
corepressor binding to BCL6, the method comprising determining
whether the test compound binds to a BCL6 lateral groove, wherein a
compound that binds to a BCL6 lateral groove inhibits corepressor
binding to BCL6.
63. The method of claim 62, wherein the test compound is an organic
compound less than 1000 molecular weight.
64. The method of claim 62, wherein the test compound is an organic
compound less than 3000 molecular weight.
65. The method of claim 62, wherein the compound is an aptamer.
66. The method of claim 62, wherein the compound is a peptide or
peptide mimetic.
67. The method of claim 66, wherein the peptide or mimetic
comprises SEQ ID NO:10.
68. The method of claim 66, wherein the peptide or mimetic
comprises SEQ ID NO:1.
69. The method of claim 66, wherein the peptide or mimetic
comprises SEQ ID NO:2.
70. The method of claim 66, wherein the peptide or mimetic
comprises SEQ ID NO:3.
71. The method of claim 62, wherein binding of the compound to the
lateral groove is determined using polypeptide comprising SEQ ID
NO:12.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/530,102, filed Dec. 16, 2003.
BACKGROUND OF THE INVENTION
[0003] (1) Field of the Invention
[0004] The present invention generally relates to inhibition of
corepressor binding to BCL6. More specifically, the invention is
directed to compositions and methods for inhibiting corepressor
binding to the BCL6 lateral groove.
[0005] (2) Description of the Related Art
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[0070] The BTB domain is a highly conserved, widely distributed
protein-protein interaction motif found in a family of
transcription factors that play critical roles in cellular
differentiation, development and neoplasia. Several BTB/zinc finger
proteins, including B-cell lymphoma 6 (BCL6) and promyelocytic
leukemia zinc finger (PLZF), are transcriptional repressors that
are implicated in human malignancy (Albagli-Curiel, 2003; Costoya
and Pandolfi, 2001; Dent et al., 2002; Lin et al., 2001; Melnick
and Licht, 1999). Both the BCL6 and PLZF proteins consist of an
N-terminal BTB domain, followed by a central region of several
hundred residues that are predicted to have little or no fixed 3D
structure, and end with a series of C.sub.2H.sub.2-type zinc finger
domains at the C-terminus. This general type of architecture is
seen in 43 of the over 200 known human BTB domain proteins (GGP and
P. J. Stogios, http://xtal.uhnres.utoronto.ca/prive/btb.html). A
second major class of BTB domain proteins contain C-terminal kelch
.beta.-propeller repeats, and many of these are thought to be
involved in cytoskeletal functions, although some of these are
involved in transcription regulation (Adams et al., 2000). The core
BTB domain fold is also found in the T1 domain of voltage-gated
K.sup.+ channels (Kreusch et al., 1998), and in the ElonginC/Slcp1
proteins (Stebbins et al., 1999).
[0071] Despite the architectural similarity of the BTB/zinc finger
transcription factors, these can function as repressors,
activators, or both and the BTB domain plays a central role in
these activities (Kaplan and Calame, 1997; Kobayashi et al., 2000;
Mahmoudi et al., 2002). The majority of BTB/zinc finger proteins,
however, are thought to be transcriptional repressors, and several
of these mediate their effects through the recruitment of histone
deacetylase complexes. Thus, in BCL6, the BTB domain mediates
interactions with the SMRT, N-CoR, BCoR and mSin3A corepressors, as
well as with histone deacetylase 1 (HDAC-1), and repression is
relieved with HDAC inhibitors (David et al., 1998; Dhordain et al.,
1997; Dhordain et al., 1998; Grignani et al., 1998; Guidez et al.,
1998; He et al., 1998; Hong et al., 1997; Huynh and Bardwell, 1998;
Huynh et al., 2000; Lin et al., 1998; Wong and Privalsky, 1998).
The recruitment of a histone deacetylase complex is not a universal
property of the BTB domain, as evidenced by the fact that the BTB
domains of HIC1 and gFBP-B do not interact with these factors
(Deltour et al., 1999). Thus, it is clear that distinct mechanisms
are used by different BTB domains in order to carry out a variety
of biological effects.
[0072] In the B-cell lineage, the BCL6 protein is expressed in
germinal center (GC) B-cells, but not in pre-B cells or in
differentiated progenies such as plasma cells. Because BCL6
expression is tightly regulated during lymphoid differentiation,
its down-regulation in post-GC B-cells may be necessary for further
plasma/memory cell differentiation. Some of the more notable genes
that are repressed by BCL6 include the B lymphocyte-induced
maturation protein (blimp-1), a transcriptional repressor of c-myc
which plays a key role in differentiation of B-cells to plasma
cells (Shaffer et al., 2002), the cell cycle control genes p27Icip1
and cyclin D2 (Shaffer et al., 2000), the programmed cell death-2
protein (PDCD2) (Baron et al., 2002), and B7-1/CD80 (Niu et al.,
2003). Chromosomal translocations upstream of the BCL6 gene are
observed in approximately 30-40% of diffuse large B-cell lymphomas
(DLBCL) and in 5-14% of follicular lymphomas (FL) (Kuppers and
Dalla-Favera, 2001; Niu, 2002; Ye, 2000). In addition, the promoter
region of BCL6 is targeted by somatic hypermutation in GC B-cells
(Pasqualucci et al., 2003; Shen et al., 1998; Wang et al., 2002).
Thus, a B-cell with an activated BCL6 gene may be trapped at the GC
stage due to the repression of differentiation and cell-cycle
control proteins (Calame et al., 2003; Dent et al., 2002; Fearon et
al., 2001; Staudt, 2002). In addition to its role in lymphoid
cells, BCL6 represses the expression of the chemokines MCP-1, MCP3
and MRP-1 in macrophages and is an important negative regulator of
TH-2 type inflammation (Toney et al., 2000).
[0073] Due to the importance of BCL6 in B-cell differentiation and
leukemia development, there is a need for greater understanding of
the mechanisms that control BCL6 interactions, particularly
interactions with the corepressors SMRT, N-CoR and BCoR. The
present invention addresses that need.
SUMMARY OF THE INVENTION
[0074] Accordingly, the invention is based on the identification of
the BCL6 site of corepressor binding, and the discovery that
peptides having the sequence of the corepressor binding site
inhibit corepressor binding to BCL6. This inhibition causes
apoptosis of B-cell lymphoma cells expressing BCL6.
[0075] Thus, in some embodiments, the invention is directed to
compounds that are capable of blocking corepressor binding to a
BCL6 lateral groove.
[0076] The invention is also directed to methods of blocking
corepressor binding to the BCL6 lateral groove binding. The methods
comprise contacting the BCL6 with any of the above-described
compounds.
[0077] The invention is additionally directed to methods of
inhibiting BCL6 repression in a mammalian cell. The methods
comprise treating the cell with any of the above-described
compounds.
[0078] In additional embodiments, the invention is directed to
methods of treating a mammal with cancer, where the cancer requires
BCL6 repression. The methods comprise administering any of the
above-described compounds, in a pharmaceutically acceptable
excipient, to the mammal.
[0079] The inventors have also identified a novel polypeptide that
is a soluble form of the BCL6 BTB domain. This polypeptide
comprises SEQ ID NO:12, which is BCL6 residues 5-129, with the
point mutations C8Q, C67R and C84N. Thus, the invention is further
directed to polypeptides comprising SEQ ID NO:12.
[0080] The invention is also directed to methods of determining
whether a test compound inhibits corepressor binding to BCL6. The
methods comprise determining whether the test compound binds to a
BCL6 lateral groove, wherein a compound that binds to a BCL6
lateral groove inhibits corepressor binding to BCL6.
BRIEF DESCRIPTION OF THE DRAWINGS
[0081] FIG. 1 is two diagrams and a graph showing the structure of
the BCL6 BTB domain. Panel A is a ribbon diagram of the BCL6 BTB
dimer (form I crystal). Panel B shows the superposition of single
chains from various BTB domains structures. In all cases, the BTB
domain forms highly similar homodimers. Panel C shows the results
of reporter assays performed in 293 T cells comparing
transcriptional repression mediated by escalating doses of vectors
expressing the GAL4 DNA binding domain fused to the BTB domain from
wild-type BCL6 (BTB.sup.BCL.6-wt), C8Q/C67R/C84N cysteine
substituted BCL6 (BTB.sup.BCL6-3C), or PLZF (BTB.sup.PLZF).
[0082] FIG. 2 is a photograph, diagrams and graphs establishing the
identification of the minimal BCL6-3C BTB interaction fragment in
SMRT. Panel A is a photograph of a stained gel of SMRT or N-CoR
fragments expressed as His-tagged thioredoxin (Trx) fusion
proteins, purified, and mixed with purified BCL6-3C BTB domain.
Complexes were affinity purified over Ni-NTA columns and separated
by SDS-PAGE. Lane a: purified Trx-N-CoR.sup.1351-1383; lane b:
purified BCL6-3C BTB domain; lanes c-j: copurifications of BCL6-3C
BTB domain with the His-tagged corepressor fusion proteins
described in panel B. Panel B is diagrams showing the "IS" and
"AAAA" substitution mutations replacing SMRT residues 1424-1425,
and 1427-1430, respectively. The open box represents the minimal
binding domain. Panel C is SPR sensograms showing the binding of
the BCL6-3C BTB domain at the indicated concentrations to
immobilized SMRT.sup.1414-1414peptide (fragment d). Panel D is a
graph of relative affinities of the BCL6-3C BTB domain for the
corepressor fragments described in panel B. The data are presented
as ratios of the dissociation constants relative to
SMRT.sup.1414-1414(fragment d), as measured by SPR. "N.b."
indicates no detectable binding. Panel E is a graph of ITC
titration of SMRT.sup.1414-1430to a solution of BCL6-3C BTB
domain.
[0083] FIG. 3 is diagrams showing the structure of the BCL6-3C BTB
domain / SMRT-BBD complex. Panel A is a ribbon diagram of the 2:2
complex. The crystallographic asymmetric unit contains the entire
four-chain structure. The N-termini of the two SMRT chains are
labeled. Panels B, C and D shows views of the BCL6-3C BTB domain
displayed as a solvent accessible surface, with the two SMRT
fragments rendered in stick representation. The two non-overlapping
surfaces of the BCL6-3C BTB dimer that are buried upon peptide
binding are the shaded areas behind the stick representations.
Panel B is a view in the same orientation as in panel A. Panel C
shows the "bottom" of the complex, viewed along the molecular
pseudo-twofold axis. Panel D shows that Ser-1424 (hidden by His-116
in this view) and Ile-1425 of SMRT are buried in a groove formed in
part by Arg-13' (.alpha.1') and His-116 (.alpha.6) from the two
chains of the BCL6-3C BTB domain. Panel E shows the sequence
alignment of selected human BTB/zinc finger proteins and the
observed secondary structure of the BCL6-3C BTB domain. The
residue-by-residue surfaces buried due to interactions with the
SMRT peptide are indicated with bars. HIC-1 has a 13 amino acid
insert at the position indicated by the three asterixes.
[0084] FIG. 4 is illustrations and a photograph showing relevant
peptide binding interactions. Panel A shows a schematic drawing of
the contacts between the BCL6-3C BTB domain and the SMRT chain.
Nearly identical contacts are observed in the other contact
surface. Panel B shows a highlight of the interactions between SMRT
1427-1430 and the BCL6-3C BTB domain. Panel C shows interactions of
SMRT 1424-1426 with the BCL6-3C BTB domain. In panel D, to view the
interactions between region 1414-1423 of the SMRT-BBD peptide and
BTB .beta.1', the BCL6-3C helix .alpha.6 has been made transparent.
Panel E shows the superposition of the two crystallographically
independent SMRT peptides from the complex. The six waters from
each site that participate in the bridging SMRT/BCL6-3C
interactions are indicated as spheres. Panel F is a photograph of a
gel showing that mutations in the BCL6-3C BTB peptide binding
pocket reduce the affinity for the SMRT peptide. His-tagged
Trx-(SMRT-BBD) was mixed with three different forms of the BCL6-3C
BTB domain, and the load ("L"), flow through ("FT"), wash ("W") and
elute ("E") fractions from each co-purification trial were analyzed
by SDS-PAGE.
[0085] FIG. 5 is graphs, photographs and micrographs showing that
the SMRT-BBD interacts with the BCL6 BTB domain in vivo. Mammalian
two hybrid assays were performed in 293 T cells by cotransfecting
the indicated expression vectors along with 100 ng of a
(GAL4).sub.5-TK-Luc reporter and 12.5 ng of a TK-renilla internal
control plasmid. Panel A is a graph of experimental results where
25 ng of bait GAL4.sup.1-147or GAL4-BCL6.sup.BTB plasmid were
transfected either alone or in combination with 300 ng of prey
VP16-(SMRT-BBD) fusions. Results are shown as relative luciferase
units to show both repression and activation. Panel B is a graph of
experimental results where 25 ng of bait plasmid expressing
GAL4.sup.1-147alone or fused to wild type or mutant BCL6 BTB
domains were cotransfected along with 300 ng of prey plasmid
expressing VP16-full-length SMRT fusions. Panel C shows GAL4
immunoblots of 293T cells transfected with 100 ng of wild-type or
mutant GAL4-BTB.sup.BCL6 plasmids. Panel D shows VP16 immunoblots
of 293T cells transfected with 300 ng of the wild type or mutant
VP16-(SMRT-BBD) plasmids. Panel E is a graph showing experimental
results where 50 ng of bait GAL4-PLZF (full-length) plasmid was
transfected alone or in combination with 200 ng of prey plasmid
expressing VP16-full-length SMRT wild type or point mutants (Top),
or 50 ng of bait GAL4-PLZF plasmid was transfected alone or in
combination with 200 or 600 ng of prey plasmid expressing
VP16-(SMRT-BBD) (Mottom). Panels F-N shows confocal laser scanning
microscopy sections collected from 293 T cells cotransfected with a
plasmid expressing full length BCL6 along with plasmids expressing
wild type (WT) or mutant (AA, IS) VP16-(SMRT-BBD) domain fusions.
Immunolocalization was performed using BCL6 monoclonal antibodies
with Cyt secondaries (F,I,L), and VP16 polyclonal antibodies with
Cy3 secondaries (G,J,M). The overlays of the BCL6 and VP16
(SMRT-BBD) images are shown in panels H,K,N.
[0086] FIG. 6 shows graphs and a photograph showing that the BCL6
BTB/SMRT-BBD interaction is critical for corepressor function.
Results are shown as fold repression compared to the relative
luciferase units of the empty vector control. Panel A is a graph
showing transcriptional repression activity of GAL4-BTB.sup.BCL6
fusions transfected with a (GAL4).sub.5-TK-Luc reporter construct.
Panel B is a graph showing results from reporter assays performed
with either full length wild-type BCL6 or full length BCL6 with the
N21K/H116A point mutations. The reporter construct
((BCL6).sub.4-TK-LUC) contains four BCL6 binding sites. Panel C is
a graph showing corepression of GAL4-BTB.sup.BCL6 fusions with
full-length SMRT. Panel D is a graph showing corepression of
GAL4-BTB.sup.BCL6 fusions with full-length BCoR. Panel E is a graph
showing the corepression effect of full-length SMRT with full
length BCL6. Panel F shows BCL6 immunoblots of 293T cells
transfected with 100 ng of wild-type and mutant full-length BCL6
plasmids. Immunoblots verifying the expression of the
GAL4-BTB.sup.BCL6 fusion proteins are shown in FIG. 5C.
[0087] FIG. 7 is diagrams, photographs and micrographs
demonstrating that BBD peptides bind to BCL6 and block recruitment
of SMRT. Panel A shows a 1.3 .ANG. resolution image of the BCL6 BTB
dimer-SMRT BBD complex. The surface of the dimer is shown in white
and the peptide backbone of each monomer is shaded. Two BBD
sequences (stick representations) bind simultaneously to the BTB
dimer. Panel B shows the amino acid sequence of the WP and MP BBD
peptides. Panel C shows immunoblots of whole cell lysates (WCL),
cytoplasm (CF) and nuclear extract (NE) 293T and Ly1 cells with HA
antibodies after a one hour exposure to PB, WP or MP. Panel D shows
immunoblots of an in vitro co-immunoprecipitation of endogenous
BCL6 with BBD peptides. Ly1 NE exposed to PB, WP or MP (0.12 mg/ml)
were precipitated with BCL6 monoclonal antibodies or normal mouse
serum (NMS) and immunoblotted with HA antibody. Panel E shows
immunoblots of in vivo co-immunoprecipitations of BCL6 and BBD
peptides. Ly1 and 293T cells transiently transfected with BCL6 were
exposed to PB or 1 .mu.M WP or MP for 1 hour. Immunoprecipitations
and immunoblotting were performed on these lysates as in panel D.
Panel F shows immunoblots of in vitro co-immunoprecipitations of
PLZF and BBD peptides. NE of 293T cells transiently transfected
with PLZF were exposed to PB or 1 .mu.M WP or MP for 1 hour. PLZF
was immunoprecipitated, and immunoblotting was performed as
indicated. Panel G shows immunoblots of in vivo
co-immunoprecipitations between BCL6 and SMRT. 293T cells were
co-transfected with BCL6 and SMRT and exposed to PB or 1 .mu.M WP
or MP for 1 hour. Lysates were subjected to immunoprecipitation
with BCL6 rabbit polyclonal antibodies or normal rabbit serum (NRS)
and immunoblotted with SMRT polyclonal antibodies. Lysate input
expression of SMRT and BCL6 and actin loading controls are also
shown. Panel H shows immunofluorescence confocal micrographs of
BCL6, SMRT, and BBD peptides. 293T cells were co-transfected with
BCL6 and SMRT and after 24 hours were exposed to PB or 1 .mu.M WP
or MP for one hour. Immunostaining was performed using mouse BCL6
with and rabbit HA or SMRT and secondary antibodies conjugated with
Cy2 (BCL6) or Cy3 (HA or SMRT), followed by DAPI staining, and
visualized by laser confocal scanning microscopy. The type of
treatment (PB, WP or MP) is indicated to the left of each row of
images.
[0088] FIG. 8 is graphs demonstrating that lateral groove
corepressor blockade is specific for BCL6 and is required for
transcriptional repression of natural and endogenous target genes.
Panels A-E shows results of reporter assays performed in 293T cells
transfected with constructs as indicated and TK-renilla as internal
control. In all cases, after 24 hours cells were exposed to PB
(white bars), or 1 .mu.M WP (gray bars) or MP (black bars) for 20
hrs and harvested for dual luciferase assays. Fold repression is
calculated compared to BCL6. Fold repression was calculated
relative to vector control for each experiment. Transfections were
performed in 24-well dishes as follows: Panel A: 50 ng of
GAL4.sup.1-147 or GAL4-BCL6.sup.BTB with 50 ng (GAL4).sub.5-TK-Luc
reporter; Panel B: 50 ng of GAL4.sup.1-147 or GAL4-HIC1.sup.BTB
with 50 ng (GAL4).sub.5-TK-Luc reporter; Panel C: 200 ng
GAL4.sup.1-147 or GAL-PLZF.sup.BTB with 50 ng (GAL4).sub.5-TK-Luc
reporter; Panel D: 50 ng of pEF vector or full length pEF-BCL6 with
50 ng (BCL6).sub.3-TK-Luc reporter; Panel E: 50 ng of pEF vector or
full length pEF-BCL6 with 50 ng (BCL6).sub.3-TK-Luc reporter alone
or with 200 ng of CMX-SMRT or 200 ng pEF-BCoR. Panel F shows the
results of real time PCR detection of mRNA of the endogenous BCL6
target genes cyclinD2, CD80, CD69 BCL6, performed in Ly1 and LY8
cells, treated with PB (white bars), or 1 .mu.M WP (gray bars) or
MP (black bars) for 7 hours.
[0089] FIG. 9 is photographs of experimental results demonstrating
that lateral groove blockade alters BCL6 target promoter
repressosome composition and chromatin structure without disrupting
DNA binding by BCL6. Panel A shows the results of electrophoretic
mobility shift assays performed using nuclear extracts prepared
from 293T cells transfected with full length BCL6. In each case 2
.mu.g of nuclear extract was allowed to interact with 10
fmol.sup.32P labeled oligonucleotides probes containing a canonical
BCL6 DNA binding site. In addition, reactions were exposed to
50-fold excess unlabeled probe in lanes 2, 9, 10, 11 and 12; 0.2
.mu.g BCL6 antibody and in lanes 3, 13 and 14, 0.1 .mu.g serum, 5
.mu.g WP in lanes 5, 9, 13 and 15; 1 .mu.g WP in lanes 6, 10, 14
and 16; 5 .mu.g MP in lanes 7 and 11; 1 .mu.g MP in lanes 8 and 12;
"X" denotes probe alone in lane 17. White arrow: high molecular
weight BCL6 mobility shift. Black arrow: lower molecular weight
BCL6 mobility shift in the presence of WP. Panel B shows the
results of chromatin immunoprecipitations (ChIP) performed in Ly1
cells treated for 7 hrs with 1 .mu.M of MP or WP. Cross-linked
chromatin was precipitated with BCL6, N-CoR, SMRT or HA polyclonal
antibodies or normal rabbit serum (NRS). The resulting purified DNA
fragments-and 5% input were amplified by end-point PCR using
primers surrounding the BCL6 binding site on the MIP-1.alpha.
promoter. Panel C shows the results of ChIP assays where Ly1 cells
were treated as above, but precipitated with histone 3, lysine
9-dimethyl (H3K9-met) or histone 4 pan-acetylated (H4-Ac)
polyclonal antibodies or NRS. The resulting purified DNA fragments
and 5% input MIP-1.alpha. promoter. Panel D shows the results of
real time PCR detection of mRNA of the endogenous BCL6 target gene
MIP1.alpha., performed in Ly1 cells treated with PB (white bars) or
1 .mu.M WP (gray bars) or MP (black bars) for 7 hours.
[0090] FIG. 10 is graphs and diagrams demonstrating that BCL6
lateral groove blockade causes apoptosis and cell cycle arrest of
BCL6 dependent human B-cell lymphoma cells. Panels A-D shows the
results of experiments where a group of cells including Ly1, Ly4,
Ly7, Ly8, Ly10, Ly12, Raji, Daudi and U937 were exposed to PB or 1
mM WP or MP for 48 hours and evaluated as follows: Panel A: cell
viability was determined by performing XTT assays. The results are
expressed relative to the viability of cells exposed to buffer
alone (which is equivalent to untreated cells); Panel B: apoptosis
was determined by FACS counting of fixed cells and stained with
propidium iodide. The fraction of apoptotic hypodiploid DNA content
(Pre-G1/G0) cells is indicated in each graph; Panel C:
Differentiation was determined by staining treated cells with
antibodies for the CD10 (germinal center) and CD38 (plasmacytic)
surface antigens and performing flow cytometry as indicated; Panel
D: Cell cycle progression was determined by fixing and staining
cells with propidium iodide and determining DNA content by FACS.
Cell cycle analysis was performed using MotFit software.
[0091] FIG. 11 shows the results of an electrophoretic mobility
shift assay for complex formation between the BCL6-3C BTB domain
and fusion proteins containing BCoR fragments. Purified BCL6-3C BTB
domain was included in all six lanes. Various fragments of BCoR as
thioredoxin-6his fusion proteins were included in lanes 1-6 as
follows: 1. BCoR 494-518; lane 2 BCoR 498-514; lane 3: BCoR
494-510; lane 4: BCoR 494-514; lane 5: BCoR 506-522; lane 6: BCoR
502-522. BCL6-3C/BCoR complexes were present in lanes 1, 2, 4 and
6.
[0092] FIG. 12 shows the results of an electrophoretic mobility
shift assay showing the effect of increasing amounts of added
BCoR-BBD peptide to the BCL6-3C BTB domain.
[0093] FIG. 13 shows the crystal structure of the BCL6-3C BTB
domain in complex with the BCoR 498-514. The BTB domain is in
Calpha representation (thin sticks), and the two BCoR peptides are
represented with the thicker sticks.
[0094] FIG. 14 shows a comparison of the crystal structures of the
BCL6-3C BTB domain in complex with the SMRT BBD (left) and the BCoR
BBD (right). The BCL6-3C BTB domain dimer is represented as a
surface, with His116 highlighted in the center of each panel, just
above the stick models. The BBD peptides are shown as stick
models.
[0095] FIG. 15 shows the superposition of the SMRT BBD and BCoR BBD
from the crystal structures.
[0096] FIG. 16 is a schematic diagram of the interactions of the
BCoR peptide with a BCL6-3C BTB dimer (chains A and B).
DETAILED DESCRIPTION OF THE INVENTION
[0097] The present invention is based on the identification of the
BCL6 site of corepressor binding, and the discovery that peptides
having the sequence of the corepressor binding site inhibit
corepressor binding to BCL6. This inhibition causes apoptosis of
B-cell lymphoma cells expressing BCL6. See Examples.
[0098] Thus, in some embodiments, the invention is directed to
compounds that bind to the BCL6 lateral groove and prevent
corepressor binding. Based on the work described in the Examples,
the skilled artisan could design many such compounds. These
embodiments are not narrowly limited to any particular compound,
and the compound can be, for example, an organic molecule less than
3000, 2000, 1500 or 1000 molecular weight, or an aptamer, both of
which can be designed or identified by known methods. In preferred
embodiments, the compound is a peptide or mimetic. These peptides
or mimetics preferably comprise the sequence xxxxzxxxxxsx(w/h)xzpx,
where x is any amino acid or mimetic analog and z is a non-polar
amino acid or mimetic analog. That sequence represents a composite
sequence of SEQ ID NO:s 1-3, which are shown in the examples to
bind to the BCL6 lateral groove, preventing corepressor
binding.
[0099] As used herein, "mimetics", also known as peptidomimetics,
includes any of the many known compounds that behave like peptides,
but are made of L-amino acid analogs that are more resistant to
degradation than peptides. Examples include peptide analogs,
pseudopeptides, depsipeptides, or, preferably, retro-inverso
peptides or mimetics of D-amino acids. Any of these peptidomimetics
to any particular peptide can be synthesized by the skilled artisan
without undue experimentation.
[0100] Numerous examples of the invention peptides or mimetics can
be identified by comparing the sequences of the BCL6 lateral groove
binding sites from the SMRT, N-CoR, and BCoR corepressors, provided
herein as SEQ ID NO:s 1-9 (see Appendix for identification of each
sequence). The 17mers identified as SEQ ID NO:s 1-3 is the minimum
sequence to achieve maximal inhibition of corepressor binding, but
larger sequences, including 84mers, or longer, are also
effective.
[0101] With the information provided in the Examples, the skilled
artisan could identify numerous peptide sequences that would bind
to the BCL6 lateral groove and inhibit corepressor binding, simply
by aligning the residues of the three corepressor sequences (SEQ ID
NO:1-3) and identifying sequences that combine the aligned
residues, or providing conservative substitutions to the amino
acids or analogs. Thus, any sequence within SEQ ID NO:10, which
represents all possible combinations of the residues of SEQ ID
NO:1-3, would be expected to inhibit binding of corepressors SMRT,
N-CoR, and BCoR. Preferably, the peptide or mimetic comprises the
sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
[0102] The peptides or mimetics can comprise any of amino acids or
analogs as the lateral groove binding moiety, up to 84 or more,
including 28 or less, 21 or less, or the minimal 17 amino acid or
analog residues. The peptide or mimetic can even include the entire
corepressor (i.e., SMRT, BCoR or N-CoR) sequence, where the
sequence is mutated to prevent the peptide or mimetic from
functioning as a corepressor.
[0103] The peptide or mimetic can also comprise one or more
functional groups, such as a moiety that facilitates purification,
e.g., a (His).sub.6 moiety or an antibody-binding epitope. Another
functional group that can be utilized as part of the peptide or
mimetic is a moiety that facilitates entry of the peptide or
mimetic into a cell, such as the protein transduction domain from
the HIV pTAT protein. An additional useful functional group here is
a moiety that facilitates detection of the peptide or mimetic, such
as a fluorescent moiety, a radioactive moiety, or an antigen. As a
preferred example of a useful peptide comprising functional
moieties, see the peptide WP, described in Example 2, which
consists of the 2 lmer having the sequence of SEQ ID NO:4, along
with a (His).sub.6 moiety, the protein transduction domain from the
HIV pTAT protein, and a hemagglutinin epitope tag for
immunodetection of the peptide.
[0104] For therapeutic uses, the peptide or mimetic is preferably
in a pharmaceutically acceptable excipient. Such compositions can
be formulated without undue experimentation for administration to a
mammal, including humans, as appropriate for the particular
application.
[0105] Additionally, proper dosages of the compositions can be
determined without undue experimentation using standard
dose-response protocols.
[0106] Accordingly, the peptide or mimetic compositions designed
for oral, lingual, sublingual, buccal and intrabuccal
administration can be made without undue experimentation by means
well known in the art, for example with an inert diluent or with an
edible carrier. The compositions may be enclosed in gelatin
capsules or compressed into tablets. For the purpose of oral
therapeutic administration, the pharmaceutical compositions of the
present invention may be incorporated with excipients and used in
the form of tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, chewing gums and the like.
[0107] Tablets, pills, capsules, troches and the like may also
contain binders, recipients, disintegrating agent, lubricants,
sweetening agents, and flavoring agents. Some examples of binders
include microcrystalline cellulose, gum tragacanth or gelatin.
Examples of excipients include starch or lactose. Some examples of
disintegrating agents include alginic acid, corn starch and the
like. Examples of lubricants include magnesium stearate or
potassium stearate. An example of a glidant is colloidal silicon
dioxide. Some examples of sweetening agents include sucrose,
saccharin and the like. Examples of flavoring agents include
peppermint, methyl salicylate, orange flavoring and the like.
Materials used in preparing these various compositions should be
pharmaceutically pure and nontoxic in the amounts used.
[0108] The peptide or mimetic compositions of the present invention
can easily be administered parenterally such as for example, by
intravenous, intramuscular, intrathecal or subcutaneous injection.
Parenteral administration can be accomplished by incorporating the
compositions of the present invention into a solution or
suspension. Such solutions or suspensions may also include sterile
diluents such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents. Parenteral formulations may also include
antibacterial agents such as for example, benzyl alcohol or methyl
parabens, antioxidants such as for example, ascorbic acid or sodium
bisulfite and chelating agents such as EDTA. Buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose may also be added. The
parenteral preparation can be enclosed in ampules, disposable
syringes or multiple dose vials made of glass or plastic.
[0109] Rectal administration includes administering the
pharmaceutical peptide or mimetic compositions into the rectum or
large intestine. This can be accomplished using suppositories or
enemas. Suppository formulations can easily be made by methods
known in the art. For example, suppository formulations can be
prepared by heating glycerin to about 120.degree. C., dissolving
the composition in the glycerin, mixing the heated glycerin after
which purified water may be added, and pouring the hot mixture into
a suppository mold.
[0110] Transdermal administration includes percutaneous absorption
of the composition through the skin. Transdermal formulations
include patches (such as the well-known nicotine patch), ointments,
creams, gels, salves and the like.
[0111] The present invention includes nasally administering to the
mammal a therapeutically effective amount of the composition. As
used herein, nasally administering or nasal administration includes
administering the composition to the mucous membranes of the nasal
passage or nasal cavity of the patient. As used herein,
pharmaceutical compositions for nasal administration of a
composition include therapeutically effective amounts of the
composition prepared by well-known methods to be administered, for
example, as a nasal spray, nasal drop, suspension, gel, ointment,
cream or powder. Administration of the peptide or mimetic
composition may also take place using a nasal tampon or nasal
sponge.
[0112] In additional embodiments, the invention is directed to
methods of blocking corepressor binding to a BCL6 lateral groove.
The methods comprise contacting the BCL6 with any of the compounds
described above.
[0113] In preferred embodiments of these methods, the BCL6 is in a
mammalian cell, preferably a cancer cell that requires BCL6
repression. Addition of the peptides to such cells cause apoptosis
in a significant percentage of the cells (Example 2).
[0114] It is preferred that cancer cells treated in these methods
are in a living mammal. The invention methods would be expected to
work in any mammal, however, in the most preferred embodiments, the
mammal is a human. Additionally, it is preferred that the cancer
cell in these embodiments is a lymphoma cell or breast cancer cell,
since those forms of cancer often require BCL6 repression to avoid
apoptosis.
[0115] It is preferred in these methods that the compound comprises
a peptide or mimetic that comprises the sequence of SEQ ID NO:10,
most preferably SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
[0116] In other embodiments, the invention is directed to methods
of inhibiting BCL6 repression in a mammalian cell. The methods
comprise treating the cell with any of the above-described
compounds. As with the methods described above, the cell is
preferably a cancer cell, most preferably a lymphoma or a breast
cancer cell. It is also preferred that the cell is in a mammal,
most preferably a human. It is also preferred that the compound
comprises a peptide or mimetic having the sequence of SEQ ID NO:1,
SEQ ID NO:2 or SEQ ID NO:3.
[0117] In related embodiments, the invention is directed to methods
of treating a mammal with cancer. The methods comprise
administering any of the above described compounds, in a
pharmaceutically acceptable excipient, to the mammal. In these
methods, the cancer requires BCL6 repression. As such, treatment
with the peptide prevents corepressor binding and causes apoptosis
of the cell.
[0118] In preferred embodiments, the mammal is a human; it is also
preferred that the cancer is a lymphoma or a breast cancer. As with
the methods described above, the preferred compound comprises a
peptide or mimetic comprising the sequence of SEQ ID NO:10, most
preferably SEQ ID NO:1, SEQ ID NO:2. or SEQ ID NO:3.
[0119] The invention is additionally directed to methods of
determining whether a test compound inhibits corepressor binding to
BCL6. The methods comprise determining whether the test compound
binds to a BCL6 lateral groove, where a compound that binds to a
BCL6 lateral groove inhibits corepressor binding to BCL6. In these
methods, the compound can be identified by any known method that
utilizes the BCL6 lateral groove structural information provided in
Examples 1 and 2.
[0120] These methods can employ a library screening protocol, i.e.,
where a library of compounds is screened for lateral groove
binding. Preferably, however, the methods utilize the structure of
the BCL6 lateral groove to aid in the design of a molecule that
would be expected to bind to the BCL6 lateral groove and inhibit
corepressor binding.
[0121] The skilled artisan could design the particular format for
these methods by utilizing known procedures, For examples, the
methods could employ an in vitro or an in vivo procedure (i.e., in
cell culture), as utilized in Examples 1 or 2.
[0122] These methods are not limited to the compound that could be
tested. The compound can, e.g., be an organic compound less than
1000, or 2000, or 3000 molecular weight. Alternatively, the
compound can be an aptamer. In preferred embodiments, the compound
is a peptide or mimetic.
[0123] The inventors have also identified a novel polypeptide that
is a soluble form of the BCL6 BTB domain. This polypeptide
comprises SEQ ID NO:12, which is BCL6 residues 5-129, with the
point mutations C8Q, C67R and C84N. Thus, the invention is further
directed to polypeptides comprising SEQ ID NO:12. As established in
Example 1, this polypeptide is very useful for testing compounds
for the ability to inhibit corepressor binding to BCL6. Also as
established in Example 1, this polypeptide can be a fusion protein
with other useful components, as described above. Also useful are
polynucleotides encoding this polypeptide, and vectors comprising
this polynucleotide.
[0124] Preferred embodiments of the invention are described in the
following examples. Other embodiments within the scope of the
claims herein will be apparent to one skilled in the art from
consideration of the specification or practice of the invention as
disclosed herein. It is intended that the specification, together
with the examples, be considered exemplary only, with the scope and
spirit of the invention being indicated by the claims which follow
the examples.
EXAMPLE 1
Mechanism of SMRT Corepressor Recruitment by the BCL6 BTB
Domain
[0125] Example Summary
[0126] BCL6 encodes a transcription factor that represses genes
necessary for the terminal differentiation of lymphocytes within
germinal centers, and the misregulated expression of this factor is
strongly implicated in several types of B-cell lymphoma. The
homodimeric BTB domain of BCL6 (also known as the POZ domain) is
required for the repression activity of the protein, and interacts
directly with the SMRT and N-CoR corepressors that are found within
large multi-protein histone deacetylase-containing complexes. We
have identified a 17 residue fragment from SMRT that binds to the
BCL6 BTB domain, and determined the crystal structure of the
complex to 2.2 .ANG.. Two SMRT fragments bind symmetrically to the
BCL6 BTB homodimer and, in combination with biochemical and in vivo
data, the structure provides insight into the basis of
transcriptional repression by this critical B-cell lymphoma
protein.
[0127] Introduction
[0128] Given the important role of BCL6 in cellular differentiation
and oncogenesis, we performed an in depth structural analysis to
determine the mechanism through which BCL6 recruits the
corepressors needed to mediate its silencing effects.
[0129] Results
[0130] BCL6 BTB domain structure. We crystallized the BTB domain
from BCL6 under two different conditions (form I and form II) and
solved its structure in the two distinct packing environments to
1.3 .ANG. and 2.2 .ANG., respectively (Table 1 and FIG. 1). It was
necessary to mutate three non-conserved cysteines to prevent
aggregation of the recombinant protein (C8Q/C67R/C84N). This
mutated BCL6 BTB domain had essentially the same activity as the
wild-type protein in transcriptional repression assays (FIG. 1C).
In all of the following sections of this example, the in vitro
experiments involving purified BCL6 BTB domain were done on the
form with the three substituted cysteines, while all cellular
assays were done in the wild-type background.
TABLE-US-00001 TABLE 1 Crystallographic statistics Form I Form I
(seMet) (native) Form II Complex Resolution (.ANG.) 2.10 2.10 2.10
1.30 2.20 2.20 Wavelength (.ANG.) 0.97947 0.97925 0.95742 0.8980
1.54 1.54 Unique Reflections 6586 6600 6719 27157 10909 16405
Redundancy 7.1 7.2 7.2 3.7 3.4 3.6 Completeness % 96.2 (73.8) 96.4
(74.8) 98.2 (87.8) 95.7 (93.7) 95.0 (85.3) 99.2 (92.1)
<|>/<.sigma.|> 21.7 (6.2) 21.7 (7.5) 24.7 (9.1) 24.4
(4.3) 12.8 (3.2) 24.0 (3.7) Rsym (%) 9.3 (23.3) 8.7 (20.6) 6.8
(17.7) 5.1 (30.2) 9.2 (28.1) 5.0 (25.6) Refinement Form I (native)
Form II Complex Resolution (.ANG.) 30.0-1.30 30.0-2.20 30.0-2.20
Space group C2 C2 P2.sub.1 Unit cell a (.ANG.) 30.61 140.75 54.23 b
(.ANG.) 71.85 32.66 38.54 c (.ANG.) 55.41 48.63 76.66 .beta.
(.degree.) 105.9 94.73 92.92 Data cutoff F/.sigma.(F) 0 0 0
R/R.sub.free, 5% (%) 12.83/17.30 20.0/24.97 22.66/26.77 RMSD bond
lengths (.ANG.) 0.0130 0.0095 0.0097 RNSD bond angles (.degree.)
1.53 1.40 1.40 Number of atoms/residues BTB.sup.BCL6 1000/122
1972/244 2002/248 SMRT-BBD 0 0 274/36 Waters 188 1130 132 Numbers
in parentheses refer to the high resolution shell (2.18-2.10 .ANG.
for Form I SeMet, 1.35-1.30 .ANG. for Form I native, 2.28-2.20
.ANG. for Form II and 2.28-2.20 .ANG. for Complex).
[0131] As expected, the BCL6 BTB domain is structurally homologous
to the PLZF BTB domain (Ahmad et al., 1998; Li et al., 1999), and
forms a tightly interwound butterfly-shaped homodimer with an
extensive hydrophobic interface. A least squares superposition of
the crystallographically unique chains of the BCL6 BTB structures
reported in this study with the BTB domain of PLZF (Ahmad et al.,
1998) reveals virtually identical structures, with an average
pairwise RMSD value of 1.0 .ANG. for equivalent C.alpha. atoms
(FIG. 1B). The N-terminus of each chain is associated with the main
body of the partner chain, generating a two-stranded antiparallel
.beta.-sheet between .beta.1 of one monomer and strand .beta.5' of
the other. The domain is an obligate homodimer, with no evidence of
exchange between subunits (KFA and GGP, unpublished
observations).
[0132] The principle dimer contacts between the BCL6 BTB subunits
are mediated by .beta.1, .alpha.2, .beta.5 and .alpha.6. Prominent
surface features of the dimer include a conserved groove formed by
the two .alpha.3/.beta.4 loops at the "top" of the dimer, and an
extensive hydrophobic concave surface formed by
.beta.1/.alpha.6'/.beta.1'/.alpha.6 on the distal "bottom" side of
the dimer.
[0133] Residues 1414-1430 of SMRT interact with the BCL6 BTB
domain. The highly related corepressors N-CoR and SMRT (also known
as N-CoR II) have an overall pairwise sequence identity of 45%, and
large segments of these proteins are predicted to be intrinsically
disordered. We have previously demonstrated the direct binding of
murine N-CoR (residues 1351-1616) and human SMRT (residues
1414-1498) to the BCL6 BTB domain (Melnick et al., 2002). A shorter
SMRT fragment from positions 1414 to 1441 binds to the BCL6 BTB
domain with similar affinity (data not shown), and we used this as
a basis for determining the minimal interaction fragment in SMRT. A
series of N and C terminal deletions were made to
SMRT.sup.1414-1441 and the binding of these fragments to the BCL6
BTB domain was assessed in a co-purification assay in which the
corepressor peptides were expressed as fusion proteins with
histidine-tagged thioredoxin (Trx) (FIG. 2A,B).
[0134] Surface plasmon resonance (SPR) biosensor measurements were
used to measure the relative strengths of interactions between the
corepressor fragments and the BCL6 BTB domain (FIG. 2C,D). We
measured a dissociation constant of 15.8.+-.3 nM for the
interaction of SMRT.sup.1414-1414 and the BCL6 BTB domain (FIG.
2C), and interestingly, a. 2.5-fold stronger affinity of the BTB
domain for the equivalent N-CoR.sup.1351-1383 peptide (FIG. 2D).
The relative binding affinities of the truncated and mutant SMRT
peptides were largely in agreement with the copurification results,
however, the SPR analysis revealed slightly weaker binding by
SMRT.sup.1417-1441 relative to SMRT.sup.1414-1414, suggesting that
residues 1414-1416 make a small but significant contribution to the
overall strength of the interaction. We observed stronger binding
of SMRT.sup.1414-1430 relative to the two longer SMRT peptides, and
this may be due to the presence of a free C-terminal carboxyl group
in this fragment. From this analysis, the minimal fragment in SMRT
required for interaction with the BCL6 BTB domain ranges from
residues Leu-1414 to Arg-1430, and we used this fragment for
further studies. We refer to this segment of the SMRT/N-CoR
corepressors as the BCL6 binding domain (BBD).
[0135] We compared the measured dissociation constant from SPR with
the value obtained in solution by isothermal titration calorimetry
(ITC) (FIG. 2E). In solution, SMRT.sup.1414-1430 (SMRT-BBD) binds
to the BCL6 BTB domain with a K.sub.d of 11.4.+-.1 .mu.M, a value
considerably less than from the SPR measurements. The stoicheometry
of the interaction by ITC is 1.11 .+-.0.06 SMRT peptides per BCL6
chain, and since the BTB domain is an obligate homodimer, these
results indicate that two peptides bind per protein dimer. There is
no indication of cooperativity. The ITC measurements reveal a
favorable enthalpic contribution (.DELTA.H=-23.2.+-.1.6 kcal/mol)
and an unfavorable entropic contribution (-T.DELTA.S=16.4.+-.2.3)
to the free energy of the interaction.
[0136] A micromolar dissociation constant is similar to the
affinity observed for SH3 (Kay et al., 2000), WW (Sudol et al.,
2001) and EVH1 (Ball et al., 2002) domains and their respective
peptide binding partners. The stronger association as measured by
SPR may be due to the fact that solid-phase techniques often
overestimate binding affinities, and differences with solution
phase measurements are often attributed to favorable avidity
effects when one component is immobilized (Ladbury et al., 1995).
Thus, for example, the affinities of the NHERF PDZ1 domain with
carboxy-terminal peptides are typically in the nanomolar range when
measured with SPR or cellular systems, but are found to be in the
micromolar range when measured in homogenous solutions by
techniques such as fluorescence polarization or ITC (Hamilton et
al., 2003). We expect that avidity effects are important in this
system because of the bivalent nature of the BCL6 BTB domain/BBD
interaction.
[0137] We failed to measure any interaction between the PLZF BTB
domain and any of the SMRT or N-CoR fragments by either the
co-purification assay, SPR or ITC. We previously showed that the
interaction of the PLZF BTB domain with the SMRT and N-CoR
fragments was at the limit of detectability by the co-purification
assay (Melnick et al., 2002), and with improvements to the method,
we now conclude that any associations between these peptides and
the PLZF BTB domain are not measurable by these techniques.
[0138] Structure of the BCL6 BTB Domain/SMRT-BBD Complex. The human
BCL6 BTB domain was co-crystallized with the SMRT-BBD peptide in a
form with an entire BTB dimer and two SMRT peptides in the
asymmetric unit. Two SMRT-BBD chains associate with the BCL6 BTB
dimer in the complex, resulting in an overall 2:2 binding ratio
(Table 1, FIGS. 3 and 4). Each corepressor fragment binds in an
extended conformation along a shallow groove formed at the BTB
dimer interface, making extensive contacts with both chains of the
BTB dimer and burying approximately 1080 .ANG..sup.2 of surface
area per peptide.
[0139] Although circular dichroism (CD) spectroscopy indicates that
the unliganded SMRT fragment is unstructured in solution, the
peptide in the complex is well defined and adopts a fixed
conformation. The two crystallographically unique SMRT chains are
virtually identical, and superpose with a Ca RMSD of 0.65 .ANG.
(FIG. 4E) and have very similar side chain conformations. There are
no SMRT/SMRT contacts, and we describe the interactions of only one
of the two corepressor chains with the BTB domain (the yellow chain
in FIGS. 3 and 4), as essentially all the interactions are
conserved across each of the two SMRT/BTB dimer interfaces.
[0140] There are only minor adjustments to the BCL6 BTB domain side
chains upon complex formation, most notably in residues Arg-13,
Arg-24, and His-116. In the unliganded structures, these amino
acids have significantly higher side chain temperature factors
relative to their neighboring residues. These adopt multiple
conformations across the form I and form II crystals, and some of
these conformers partially block the ligand binding groove.
However, in the SMRT complex, these three residues are
well-structured and make some of the more important ligand contacts
(FIG. 3E). For example, in order to accommodate the SMRT fragment,
Arg-13 swings out of the ligand binding groove, forms a hydrogen
bond with Asp-17 from the same BCL6 chain, and makes numerous polar
and non-polar contacts with the peptide.
[0141] The main chain torsion angles of the corepressor peptide all
lie within the .beta.-strand region of the Ramachandran plot, with
the exception of Gly-1422 and Ser-1424, which are in the
.alpha.-helical region. This causes a kink in the middle of the
peptide, allowing the C-terminal half of the fragment to run up the
front of the BTB dimer. Residues from both BTB chains contact each
of the SMRT peptides, with contributions mainly from .beta.1' and
.alpha.1' from one BCL6 BTB chain, and .alpha.2, .alpha.3 and
.alpha.6 from the other. The peptide binding interface is mostly
polar, and the majority of protein-peptide interactions are
mediated through backbone and sidechain hydrogen bonds, as well as
through water-mediated hydrogen bonds. Both sites contain six
bridging waters that contact both the peptide and the BTB domain
(FIG. 4E), and three of these waters are also found in the
unliganded BCL6 BTB crystals.
[0142] The N-terminal portion of each SMRT peptide interacts in
parallel with BCL6 .beta.1, and adds an additional .beta.-strand to
the existing .beta.1/.beta.5' sheets at the bottom of the dimer
(FIGS. 3 and 4D). The majority of the interactions in this region
are mediated by main-chain hydrogen bonds between the peptide and
the amino terminus of one of the BCL6 BTB chains. The relevance of
these interactions is supported by the binding data, as SMRT
residues 1414-1416 make only minor contributions to the strength of
the interaction, while the deletion of residues 1414-1420 abrogates
complex formation altogether (FIG. 2).
[0143] In the middle region of the peptide (FIG. 4C), SMRT residues
Ser-1424 and Ile-1425 are deeply buried in the complex, and are
found in a groove in the BCL6 BTB domain bounded by Arg-13'
(.alpha.1') and His-116 (.alpha.6). Ser-1424 makes three hydrogen
bond interactions to the BCL6 BTB domain, and Ile-1425 points in
towards a hydrophobic pocket formed in part by Val-18 (.alpha.1')
and Cys 53 (.alpha.2). As expected, the SI to IS mutation abrogates
binding of the SMRT-BBD to the BCL6 BTB domain (FIG. 2), indicating
that these residues make critical contributions to complex
stability. Surprisingly, BCL6 residues Arg-13 and His- 116 make
some of the largest contributions to the buried interface surface
in the complex, yet they are not conserved within the BTB domain
family (FIG. 3E).
[0144] A network of polar and non-polar interactions between the
C-terminal end of the SMRT peptide and the second half of helix al
also contribute to the overall stability of the complex (FIG. 4D),
although the contacts in this region are not as extensive as in
other parts of the interface.
[0145] This region is nonetheless required for binding, since
replacing residues 1427-1430 of SMRT-BBD with alanines
(EIPR->AAAA) abolished the binding as measured by SPR and the
copurification assay (FIG. 2). Of these four residues, Ile-1428
makes the most contacts with the dimer.
[0146] Mutations in the BCL6 BTB domain abrogate the interaction
with SMRT-BBD. To further validate the interaction, we mutated BCL6
residues that contact the SMRT peptide. The H116A mutation has a
significantly reduced affinity for Trx-(SMRT-BBD), while the N21K
mutation showed no binding at all (FIG. 4F). The latter mutant was
chosen to introduce the equivalent residue from PLZF into BCL6
(FIG. 3E), and was predicted to be incompatible with SMRT binding
based on the observed structure of the complex. The H116A and N21K
mutants were well expressed as soluble proteins in E. coli, and the
purified proteins eluted as single peaks by gel filtration
chromatography at similar elution volumes as the native protein. In
addition, the two mutant proteins had nearly equivalent CD spectra
as the wild type protein, and all three proteins.sup.-had similar
thermal denaturation midpoint transition temperatures
(60.2.degree., 63.1.degree. and 61.0.degree. for the wt, H116A and
N21K mutants, respectively). Thus, any differences in the
biochemical and biological activity of these mutants are most
likely due to changes in the protein-protein interaction properties
of the domain, and not due to non-specific effects such as defects
in folding (Melnick et al., 2000).
[0147] SMRT BBD interacts with the BTB domain of BCL6 but not PLZF
in vivo. We next tested whether the interaction of the SMRT-BBD
with the BCL6 BTB domain occurred in a similar manner in vivo in
mammalian two-hybrid assays. Previous work showed that the
interaction of the BCL6 BTB domain with SMRT can be detected in
such experiments (Huynh and Bardwell, 1998; Melnick et al., 2002).
GAL4-BTB.sup.BCL6 was co-expressed and allowed to interact with
wild-type or mutant VP16-(SMRT-BBD) peptide fusions with
substitutions at the critical Ser-1424 and Ile-1425 positions (FIG.
5A). In the absence of the SMRT prey, GAL4-BTB.sup.BCL6 repressed
transcription from the (GAL4).sub.5-TK-Luc containing reporter
construct. When VP16-(SMRT-BBD) was co-transfected with GAL4-
BTB.sup.BCL6, the transcriptional response switched from repression
to activation, indicating that the two proteins interact. In
contrast, both SMRT mutants were unable to mediate activation,
indicating weak or no binding to the BTB domain of BCL6. We next
performed reciprocal experiments in which GAL4-BTB.sup.BCL6
wildtype and mutant fusion proteins was used as the bait to capture
VP16 activation domain fusions with full-length SMRT (FIG. 5B). The
N21K, H116A and N21K/H116A BTB mutants gave background signals
indicating no interaction, while the wild-type BCL6 BTB domain
produced an activation signal in this assay, indicating that the
two proteins interact. Equivalent expression levels of the
constructs were verified by immunoblotting (FIG. 5C and D).
[0148] We previously reported that the interaction between the PLZF
BTB domain and SMRT was undetectable by mammalian two-hybrid assays
(Melnick et al., 2002). However, a full-length PLZF GAL4 fusion was
able to generate a mammalian two-hybrid signal when co-expressed
with full-length VP16-SMRT (FIG. 5E). The interaction was
unaffected when SMRT residues 1424 and 1425 were mutated to either
AA or IS. In addition, no interaction was observed when
VP16-(SMRT-BBD) was used as the PLZF prey. Therefore, the
interaction of the SMRT BBD motif described here is specific for
BCL6 and not PLZF both in vivo and in vitro. Interestingly, the
BCL6 BTB domain is a much more potent transcriptional repressor
than the PLZF BTB domain when equivalent amounts of GAL4-BTB
expression plasmid are used in reporter assay (FIG. 1 C),
correlating with the significantly greater in vitro and in vivo
affinity for SMRT.
[0149] BCL6 binding to SMRT-BBD directs localization to nuclear
speckles. BCL6 normally localizes to nuclear speckles in a
BTB-dependent manner (Dhordain et al., 1995). Furthermore, BCL6 and
SMRT colocalize in nuclear speckles when overexpressed in
transfected cells (Huynh and Bardwell, 1998). Full-length BCL6
could recruit VP16-(SMRT-BBD) to nuclear speckles, while the
VP16-(SMRT-BBD.sup.SI->AA) and VP16-(SMRT-BBD.sup.SI->IS)
mutants showed virtually no spatial overlap with BCL6 (FIG. 5F-N).
The BBD motif is therefore sufficient to direct recruitment to BCL6
nuclear speckles in vivo.
[0150] The BTB lateral groove is required for BCL6 transcriptional
repression. Since the BCL6 lateral groove appears to the major
binding site for BBD containing corepressors, we tested whether
this contact was required for transcriptional repression by the
BCL6 BTB domain. GAL4-BTB.sup.BCL6 wild-type or mutant constructs
containing the N21K, H116A or N21K/H116A point mutation(s) were
co-transfected with a (GAL4)5-TK-Luc reporter. In contrast to
wild-type BCL6 BTB, these mutants were completely unable to repress
transcription regardless of the dose of expression plasmid
administered, suggesting that corepressor binding in this site
mediates BTB dependent transcriptional repression (FIG. 6A). When
these same mutations were introduced into full-length BCL6,
repression activity was greatly reduced, though not abrogated,
indicating that motifs in other regions of BCL6 may partially
compensate for the defect in BTB corepressor recruitment (FIG. 6B)
(Lemercier et al., 2002; Zhang et al., 2001).
[0151] The BCL6 BTB lateral groove motif is required for functional
interaction with corepressors. Functionally, SMRT, N-CoR and BCoR
enhance the levels of repression by BCL6 (Dhordain et al., 1997;
Huynh and Bardwell, 1998; Huynh et al., 2000). The interactions
between the BCL6 BTB domain and SMRT, N-CoR and BCoR are mutually
exclusive (Huynh et al., 2000), and these corepressors presumably
compete for a common binding site on the BTB domain. BCoR contains
the sequence motif EIPK from residues 481-484, which may be related
to the EIPR portion of the SMRT-BBD motif described here. Wild-type
and mutant GAL4-BTB.sup.BCL6 fusion proteins were co-expressed with
corepressors in reporter assays. SMRT and BCoR enhanced wild-type
BTB repression by 2-3 fold but did not significantly enhance
repression by the mutant BTB domains above control levels,
indicating that interactions with the BCL6 BTB lateral groove are
required for the effect of these corepressors with the BTB domain
of BCL6 (FIG. 6C and D).
[0152] Finally, to determine whether the BTB lateral groove was
also required for corepression of full length BCL6, we tested
whether full-length BCL6 harboring the N21K/H116A mutations could
functionally interact with SMRT (FIG. 6E and F). While SMRT
enhances repression by wild-type full-length BCL6, the protein
containing the N21K/H116A mutations is unaffected by the presence
of SMRT. Therefore, the lateral groove of the BCL6 BTB domain is
required for both physical and functional interaction between BCL6
and BBD-containing corepressors.
[0153] Discussion
[0154] The BTB domain of BCL6 may have two roles: first, the
dimerization and possible oligomerization of the domain is an
architectural feature necessary for the normal function of the
protein. Second, direct interactions with corepressors are required
for the BTB-mediated repression effects. The BCL6 BTB dimer can
bind two BBD peptides, and in a biological context, avidity effects
may be important for the association of a BCL6 to HDAC complexes
that contain two or more SMRT, N-CoR and/or BCoR chains.
[0155] Previous observations of dimer-dimer associations between
the .beta.1 regions of PLZF BTB dimers (Ahmad et al., 1998; Li et
al., 1999) support the suggestion that higher-order BTB complexes
may be functionally important (Ball et al., 1999). We observe only
one such interaction between BCL6 dimers in the form I and form II
structures presented here out of three possible. If similar BTB
dimer-dimer associations are important for BCL6 in vivo, it is
likely that the association of the SMRT peptide would disrupt these
assemblies. Further study will be required to clarify the role, if
any, of these effects.
[0156] In addition to SMRT, N-CoR and BCoR, the BCL6 BTB domain has
been shown to interact with mSin3A and histone deacetylase 1 (David
et al., 1998; Dhordain et al., 1998; Wong and Privalsky, 1998),
suggesting that several distinct contacts may occur between a BTB
domain transcription factor and the components of large repression
complexes. While the BTB lateral groove is the site of binding for
the BBD motif, additional proteins may recognize other surface
features of the domain. The sequences of BTB domains are very
diverse (for example, the PLZF and BCL6 BTB domains share only 28%
sequence identity), and it is possible that there are additional
protein-protein recognition modes in this domain family. Many other
protein interaction domains display a wide range of ligand binding
properties (Pawson and Nash, 2003). For example, the PH, PTB and
EVH1 domains share a common core fold, yet these bind a large
variety of peptide or phospholipids ligands using distinct binding
sites distributed across the domain surface (Prehoda et al., 1999).
In particular, the conserved charged groove at the top of the BTB
dimer is a possible protein-protein interaction site (Ahmad et al.,
1998; Melnick et al., 2000; Melnick et al., 2002).
[0157] The specificity of the SMRT-BBD for the BCL6 BTB domain but
not the PLZF BTB domain correlates with the relative strengths of
the domains as transcriptional repressors (FIG. 1C; Melnick et al.,
2002). An examination of the BCL6 residues that contact the
SMRT-BBD peptide provides some insight into the large difference in
affinity between the two BTB domains.
[0158] Out of the approximately 30 residues of the BCL6 dimer that
are buried upon complex formation with SMRT, only three positions
(His-14, Asn-23 and Lys-123) are identical in PLZF, while 7 more
positions are similar (FIG. 3E). Furthermore, side-chains that make
sizable contributions to the buried interface surface in the
complex, such as Arg-13, Asn-21, Arg-24, Arg-28 and His-116, are
not conserved between the two proteins. An analysis of the
corresponding residues in ZID (Bardwell and Treisman, 1994),
another transcriptional repressor that recruits components of the
histone deacetylase complex (Huynh and Bardwell, 1998), also
indicates that the majority of the residues that make sizable
contributions to the BCL6/SMRT-BBD complex are not conserved.
[0159] The apparent unique specificity of the BCL6 BTB domain for
the BBD motif makes this an attractive system for the design of
small molecule inhibitors. Recognition of the corepressor motif is
essential for the transcriptional repression activity of BCL6, and
compounds that disrupt this interaction have potential as
therapeutic agents for BCL6-related B-cell lymphoma. Such
inhibitors would release the differentiation block in these
lymphocytes in a fashion similar to the use of retinoic acid in
t(15:17) acute promyelocytic leukemia (APL) (Costoya and Pandolfi,
2001; Lin et al., 2001; Zelent et al., 2001).
[0160] Experimental Procedures
[0161] Plasmids. Fragments of human BCL6 (codons 5-129), SMRT or
N-CoR were subcloned into a modified pET-32 expression vector
(Novagen), encoding a thioredoxin fusion protein containing a 6-His
tag, a TEV protease site, a BirA biotinylation recognition motif
(Cull and Schatz, 2000) and a thrombin protease site N-terminal to
the insert site. The GAL4-BTB.sup.BCL6, BTB.sup.PLZF and PLZF
mammalian expression vectors, and the (GAL4).sub.5-TK-Luc reporter
constructs were previously described (Li et al., 1997; Melnick et
al., 2002). VP16-full length SMRT fusions, full-length BCL6
expression plasmid, BCoR expression plasmid and BCL6 binding site
reporters were a gift of V. Bardwell (University of Minnesota)
(Huynh and Bardwell, 1998; Huynh et al., 2000). Point mutations
were introduced using the QuikChange reagents and protocols
(Stratagene).
[0162] Protein Expression and Purification. Fusion proteins were
expressed in E. coli BL21(DE3), and were purified by Ni-NTA
affinity chromatography (Qiagen) followed by Superdex-75
chromatography (Pharmacia) in 250 mM NaCl, 20 mM Tris-HC1, pH 8.0.
BCL6 BTB domain protein was produced by thrombin digestion of the
thioredoxin fusion proteins, and repurified by size-exclusion
chromatography in 500 mM NaC1, 20 mM Tris-HCl, 10% glycerol, 1.0 mM
TCEP, pH 8.5. Corepressor fusion proteins were biotinylated with
BirA and digested with TEV protease to yield peptides for SPR
analysis with a biotinylated lysine eight amino acids N-terminal to
the first corepressor residue, or digested with thrombin to produce
peptides with no additional N-terminal residues for
co-crystallization or ITC.
[0163] In vitro binding assays. Co-purification: Equimolar amounts
of His-tagged thioredoxin-corepressor fusion protein and BCL6 BTB
or PLZF BTB domain were combined, incubated for at least two hours,
and loaded onto a Ni-NTA spin column (Qiagen). The column was
washed three times with 600 .mu.l of wash buffer (200 mM NaCl, 20
mM Tris-HCl pH 8.0, 10 mM imidazole), and bound protein was
released with 400 .mu.l of elution buffer (200 mM NaCl, 20 mM
Tris-HCl pH 8.0, 300 mM imidazole). Samples were analyzed on 10-20%
SDS-PAGE gels and stained with Coomassie blue.
[0164] SPR: Biotinylated corepressor peptides were coupled to
streptavidin-coated sensor chips to a density of 800 response units
(Biacore 2000). BCL6 or PLZF BTB protein was serially diluted in
running buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005%
v/v surfactant P20) and injected at a flow rate of 5 .mu.L/min.
[0165] ITC: The binding of SMRT.sup.1414-1430 to BTB domain protein
was analyzed on a Microcal VP-ITC instrument at 25.degree. C. All
components were in 150 mM NaCl, 20 mM Hepes pH 7.7, 1 mM TCEP. The
experiment consisted of thirty 6.93 .mu.L injections of a solution
containing 480 .mu.M SMRT.sup.1414-1430 into a sample cell
containing 1.8 mL of either BCL6 BTB (23.0 .mu.M) or PLZF BTB (38.5
.mu.M). To correct for dilution and mixing effects, a series of
control injections was carried out, in which the heat of dilution
was measured in blank titrations by injecting the peptide into the
buffer.
[0166] Crystallization and Structure Determination. Form I
BTB.sup.BCL6 : Crystals were grown at room temperature in hanging
drops by mixing 2 .mu.L of 8 mg/mL protein with 2 .mu.L of
reservoir buffer (0.18 M sodium formate and 0.1 M sodium acetate,
pH 4.8). A native dataset to 1.3 .ANG., and three wavelength
anomalous diffraction dataset on selenomethionine-substituted
protein to 2.1 .ANG. were collected at 100 K at beamline 14D of the
Advanced Photon Source (APS). The data were processed with DENZO
(Otwinowski and Minor, 1997). SOLVE was used for locating the
selenium atoms and initial phasing (Terwilliger and Berendzen,
1999). Initial model refinement was with CNS (Brunger et al.,
1998), and later continued with SHELXL-97 (Scheldrick and
Schneider, 1997) on the native dataset.
[0167] Form II BTB.sup.BCL6: Crystals were grown at room
temperature in hanging drops by mixing 2 .mu.L of protein with 2
.mu.L of reservoir buffer (30% PEG 8000, 0.18 M Sodium Acetate, 0.1
M HEPES pH 7.0). Data were collected at 100 K on a MAR-345 Imaging
Plate Detector on a Rigaku RU200 with a copper target. The
structure was solved by molecular replacement (CNS) using the form
I BCL6 BTB structure, and refined with CNS.
[0168] BTB.sup.BCL6/SMRT.sup.1414-1430 complex: Crystals were grown
in hanging drops by mixing 2 .mu.L of a protein solution containing
15 mg/mL BCL6 BTB and a 2.5 molar excess of SMRT.sup.1414-1430 with
2 .mu.L of reservoir buffer (25% PEG 3350 and 0.2 M ammonium
acetate). Data collection, structure solution and refinement were
as described for the form II crystals. Molecular graphics in FIGS.
1, 3 and 4 were generated with PyMol, Molscript and Raster 3D.
[0169] Mammalian two hybrid assays. Assays were performed in
5.times.10.sup.5293 T cells plated in 12-well dishes. GAL4 fusion
expression "bait" vectors and VP16 "prey" vectors were
cotransfected as indicated in the figure legends. Transfections
were performed in quadruplicate using the Superfect lipid reagent
(Qiagen) and were repeated 4 to 8 times. Cell lysates were
subjected to dual luciferase assays (Promega). Equivalent levels of
protein expression from the transfected plasmids was verified by
immunoblotting with rabbit polyclonal GAL4.sup.1-147 (sc-577)
antibodies or mouse monoclonal VP16 (14-5) antibodies (Santa
Cruz).
[0170] Immunofluorescence. 293 T cells were transfected with either
100 ng of pEF-BCL6 or pEF vector control with 200 ng VP16-SMRT
constructs or VP16 vector plasmid control. Cells were fixed in ice
cold methanol, blocked with 10% donkey serum, permeabilized in 0.1%
Tween and then exposed to BCL6 D-8 monoclonal antibody (Santa Cruz)
and VP16 polyclonal antibody (Clontech). The cells were exposed to
donkey anti-mouse secondary antibodies conjugated to Cy2 and donkey
anti-rabbit secondary antibodies conjugated to Cy3 (Jackson
Immuno-Research, West Grove, Pa.). Vectashield mounting medium with
4', 6'-diamidino-2-phenylindol (DAPI) was then applied (Vector
Laboratories, Burlington Calif.). Images were collected using a
BioRad Radiance 2000 Laser Scanning Confocal Microscope.
[0171] Repression assays. Reporter assays were performed in 293 T
cells seeded at a density of 5.times.10.sup.5 cells per well of a
twelve well dish. 100 ng of either (GAL4)-TK-Luc and (BCL6)4-
TK-Luc reporters were co-transfected with 10 ng of TK-Renilla
internal control plasmid using Superfect (Quiagen). Lysates were
submitted to dual luciferase assays as per the manufacturer's
protocol (Promega). Equivalent levels of protein expression from
the transfected plasmids was verified by immunoblotting with rabbit
polyclonal GAL4.sup.1-147 (sc-577) antibodies or mouse monoclonal
BCL6 (D8) antibodies (Santa Cruz).
[0172] Accession Numbers. The coordinates for the form I, form II
and BBD peptide complex crystal structures of the BCL6 BTB domain
have been deposited to the Protein Data Bank with accession codes
1R29, 1R28 and 1R2B, respectively.
EXAMPLE 2
Dissecting the BCL6 Repressosome in vivo as Transcription Therapy
for B-cell Lymphomas
[0173] Example Summary
[0174] The BTB/POZ transcriptional repressor BCL6 is frequently
misregulated in B-cell lymphomas. We identified the interface
through which the BCL6 BTB domain mediates recruitment of the SMRT,
N-CoR and BCoR corepressors. To determine the contribution of this
interaction to BCL6 mediated gene silencing and lymphomagenesis we
generated specific peptide inhibitors that penetrate cells, bind
BCL6 and block corepressor recruitment. These peptides modified the
chromatin structure of BCL6 target promoters, abrogated BCL6
mediated repression, reactivated BCL6 target genes, and induced
apoptosis and cell cycle arrest in B-cell lymphoma cells.
Therefore, SMRT, N-CoR and BCoR play essential roles in BCL6
repression and are required for BCL6 to maintain the malignant
phenotype of diffuse large B-cell lymphoma cells. BCL6 BTB blockade
may thus constitute a novel form of targeted transcription
therapy.
[0175] Research Rationale, Results and Discussion
[0176] The BTB/POZ domain is a highly conserved .about.120 residue
polypeptide motif found in over 200 human proteins. A subfamily of
BTB proteins contain C-terminal DNA binding motifs (usually C2H2
zinc fingers) and function as transcriptional repressors, several
of which are implicated in human cancers including the BCL6, PLZF
and Hic-1 proteins. In all of these proteins, the BTB domain is
required for transcriptional repression, dimerization,
oligomerization, and localization to specific nuclear compartments.
We are interested in the mechanisms of action of these
transcriptional repressors and the role of the BTB domain in the
molecular pathophysiology of human cancer. We previously showed
that BTB domains mediate obligate homo-dimerization through a
conserved hydrophobic face and oligomerization through a separate
hydrophobic surface region. These architectural features are
conserved and swappable among BTB proteins and required for their
biological functions.
[0177] BTB domains also mediate repression through actions specific
to each protein. In particular, the BCL6 BTB domain is a potent
repressor that directly binds SMRT, N-CoR and BCoR corepressors in
a mutually exclusive manner. Mapping experiments indicated that
these corepressors bind the BCL6 BTB domain through a conserved 17
residue motif (hereon called "BBD", for BCL6 binding domain).
Crystallographic analysis of the BCL6BTB/BBD complex revealed that
the BBD directly binds a "lateral groove" motif specific to the
BCL6 BTB domain (FIG. 7A). The lateral groove-BBD interaction plays
a key role in BCL6 interaction with SMRT, N-CoR and BCoR in vivo
and is required for BTB domain-mediated transcriptional repression
of reporter constructs.
[0178] Transcriptional repression of target genes is the only known
action of BCL6, and is attributed in large part to the BTB domain.
However, the BCL6 ZnFs participate in repression as well by
recruiting the ETO corepressor and class II HDACs and a medial
region of BCL6 also has repressor activity. In addition to
repression through direct binding to target genes, BCL6 may
function as a corepressor for AP-1 in a BTB domain-dependent manner
and to reduce GATA-3 protein levels through a post-transcriptional
mechanism. The relevance and hierarchy of each of these domains and
mechanisms of BCL6 gene regulation are unknown. Finally, little is
known of what contributions are made to endogenous transcriptional
repression by SMRT/N-CoR and BCoR and how this impacts on the
biological actions of their partner transcription factors.
[0179] Regulatory elements of the BCL6 gene are frequently mutated
in human diffuse large B-cell lymphomas (DLBCL) by chromosomal
translocations or somatic hypermutation. This leads to
inappropriately timed expression of BCL6, which is otherwise
tightly controlled in B-cells. Normally, BCL6 detains
antigen-stimulated B-cells in lymphoid follicles to form germinal
centers and permits maturation of activated B-cells to
high-affinity immunospecific lymphocytes. In lymphomas, sustained
BCL6 expression is postulated to favor B-cell proliferation and
survival in the face of ongoing mutagenesis by the somatic
hypermutation machinery. In spite of intensive studies, and its
consideration as a molecular marker of disease prognosis in B-cell
lymphomas, it is not known whether BCL6 is required to maintain the
malignant phenotype of tumor cells and whether such a role is
restricted to cells with mutations in the BCL 6 gene (40% of
cases), or to all BCL6 positive B-cell lymphomas (80% of
cases).
[0180] Since our previous data (Example 1) indicate that the
lateral groove-BBD interaction is a critical interface for
SMRT/N-CoR/BCoR recruitment to the BCL6 BTB domain, we reasoned
that lateral groove blockade offers the unique possibility of
determining the contribution of these corepressors to
transcriptional and biological actions of BCL6, the contribution of
BCL6 repression to its biological actions and, the contribution of
BCL6 to the malignant phenotype of different classes of B-cell
lymphomas. Finally, were BCL6 to require lateral groove corepressor
recruitment for oncogenic effects, blocking reagents would
constitute a highly specific and potent form of transcription
therapy for human patients with these diseases.
[0181] To test this hypothesis, a peptide inhibitor (WP) was
designed (FIG. 7B), consisting of four basic elements: an
N-terminal (His)6 tag for affinity purification, a protein
transduction domain (PTD) from the HW pTAT protein for peptide
delivery into the cells (Frankel and Pabo, 1988), a hemagglutinin
(HA) epitope tag for immunodetection, and the human SMRT BBD
(residues 1414-1430), for specific binding to the BCL6 lateral
groove (FIG. 7B). As a negative control a mutant peptide (MP) was
engineered where the BBD contained an EIPR->AAAA mutation, which
we previously showed (Example 1) abrogates binding to BCL6. These
peptides were expressed in E. coli and purified from the insoluble
fraction of bacterial lysates.
[0182] To verify transduction and intracellular localization,
B-cell lymphoma cells (Ly1, Ly4 and Ly8 DLBCL cells, Raji and Daudi
Burkitt lymphoma cells) and 293T cells were treated with several
different concentrations of purified WP and MP. Whole cell lysates,
cytoplasmic and nuclear extracts were obtained and western blots
performed with .alpha.-HA antibodies to detect the peptides (FIG.
7C). Both WP and MP peptides efficiently penetrated cells and
localize preferentially in the nucleus. We previously showed that
the BBD binds to BCL6 but not other BTB proteins. To confirm
specific binding of our peptides to BCL6, Ly1 nuclear extracts were
mixed with peptide buffer (CB), or 1 .mu.M of WP or MP
respectively. Ly1 cells are used extensively in these studies,
since they are derived from a DLBCL patient with BCL6 gene exon 1
mutations that cause loss of BCL6 autoregulation--and are thus a
potentially "BCL6 dependent" tumor.
[0183] Co-immunoprecipitations (Co-IP) using anti-BCL6 antibodies
were performed followed by western blot against the HA epitope of
the peptides (FIG. 7D). Consistent with our expectations, WP but
not MP interacted with endogenous BCL6 from Ly1 cells. Similar
results were obtained using Lysates from 293T cells transfected
with full-length BCL6 and exposed to BP or 1 .mu.M WP or MP for one
hour (FIG. 7E).
[0184] We next determined whether lateral groove binding by WP
peptide could displace the SMRT corepressor in vivo.
Co-immunoprecipitations between SMRT or N-CoR with BCL6 are
challenging with existing antibodies and hence there are no
examples of such experiments in the literature. Accordingly, we
found that coimmunoprecipitation of BCL6 with endogenous
corepressors cells was elusive since these proteins are expressed
at low levels. However, by extensive optimization we obtained
reproducible co-immunoprecipitations between BCL6 and SMRT in
transfected 293T cells. Forty-eight hours after transfection, the
cells were exposed to either PB, or 1 .mu.M WP or MP for one hour.
Remarkably, the WP peptide consistently abrogated SMRT binding to
BCL6, whereas the mutant peptide failed to do so (FIG. 7F). BCL6
normally localizes in nuclear speckles, where it colocalizes with
SMRT. By transfecting low levels of BCL6 and SMRT in 293T cells we
were able to reproduce the endogenous staining pattern of BCL6 and
SMRT. The impact of lateral groove blockade on BCL6-SMRT
colocalization was determined by exposing these cells to PB, WP or
MP as described above. Cells were then co-stained for BCL6 and SMRT
or BCL6 and HA and visualized in sections by confocal microscopy
(FIG. 7G). We found that BCL6 and SMRT colocalization occurred in
the presence of PB or 1 .mu.M MP, but was disrupted in presence of
1 .mu.M WP. Reciprocally, BCL6 colocalized with WP but not MP,
indicating that WP peptide binding was mutually exclusive with
corepressor binding, and that the BTB lateral groove contact site
is necessary and sufficient for SMRT binding to BCL6 in vivo.
Furthermore, structure guided design of blocking molecules can lead
to production of effective inhibitors of transcription
factor-corepressor interactions proteins.
[0185] Our previous data indicate that lateral groove point
mutations disrupt transcriptional repression by the BCL6 BTB
domain, consistent with loss of corepressor recruitment. We
reasoned that since WP peptides block SMRT binding to BCL6, BTB
domain-mediated transcription would be impaired. To determine
whether this is the case, 293T cells were transfected with a
GAL4-BCL6.sup.BTB fusion expression vector and a GAL4 responsive
reporter construct and an internal control reporter. Twenty four
hours after transfection, cells were exposed to PB, WP, MP or
nothing over the next 20 hours. Remarkably, WP but not MP or PB
abrogated BCL6 BTB domain transcriptional repression (FIG. 8A).
Growth and viability of 293T cells was unaffected by any of these
reagents and the protein levels of GAL4BCL6.sup.BTB fusions was
equivalent in all samples (data not shown).
[0186] Other BTB domains autonomously repress transcription, yet
none appear to contain lateral groove motifs. For example, the PLZF
BTB domain also directly interacts with and requires SMRT and
N-CoR, though interaction seems to occur through a distinct charged
pocket motif, while the HIC-1 BTB domain also mediates repression
but does not interact with SMRT or N-CoR. Therefore,
transcriptional repression by these BTB domains should not be
affected by the WP peptide. Accordingly, in contrast to BCL6,
transcriptional repression by the GAL4-PLZF.sup.BTB or GAL4-HIC1
proteins were unaffected by WP or negative controls (FIG. 8A).
These results suggest that the lateral groove--BBD mechanism is
specific to BCL6 and not other BTB proteins, consistent with our
bio-informatic predictions.
[0187] We next wished to determine the hierarchy of the lateral
groove mechanism, i.e. the contribution of the BBD corepressors, to
transcriptional repression by full-length BCL6. Full length BCL6
was expressed together with a (BCL6).sub.3 binding site-TK-Luc
reporter and then exposed to increasing doses of WP or MP peptides
(250 nM, 500 nM and 1 .mu.M) followed by peptide treatment as
above. WP but not MP caused a dose-dependent reduction of BCL6
transcriptional repression, but did not completely abrogate it
(FIG. 8B), suggesting that the remaining BCL6 domains mediate
residual repression independent of SMRT/N-CoR/BCoR. Alternatively,
loss of BCL6 repression may be underestimated by the lag time
between transfection and peptide exposure. Finally, the
SMRT/N-CoR/BCoR corepressors normally enhance transcriptional
repression by BCL6 in reporter assays. Not surprisingly, lateral
groove blockade with WP inhibits enhancement of BCL6 repression by
BCoR and SMRT (FIG. 8C).
[0188] Transcriptional regulation differs in episomal reporter
genes vs. the structured chromatin context of endogenous gene loci.
The most significant measure of lateral groove effect is thus
whether peptide blockade of endogenous BCL6 re-activates endogenous
direct BCL6 target genes. We exposed three DLBCL cell lines: Ly1
(mutated BCL6 exon 1), Ly8 (BCL6 translocation), Ly4 (Non mutated
BCL6 locus) to buffer, or 1 .mu.M WP or MP for 6 hours, extracted
mRNA and performed real time PCR on BCL6 target genes. WP peptide
re-activated BCL6 target genes such as CD80 5-8 fold and cyclinD2
2-4 fold in Ly1 and Ly8 cells, whereas there was no effect in Ly4
cells (FIG. 8D). In contrast, MP had little effect on BCL6 target
gene expression. Although BCL6 autoregulates its own expression,
there was no detectable re-activation in these lymphoma cells, most
likely since Ly1 and Ly8 have lost the BCL6 regulatory elements in
the mutated alleles. These studies offer direct evidence that the
BBD proteins function as corepressors in vivo, and their
interaction with the BTB domain is required to maintain silencing
by the endogenous BCL6 protein.
[0189] To determine whether the effects of WP peptide on repression
might be caused by interfering with BCL6 binding to DNA, we next
performed electrophoretic mobility assays. Nuclear extracts of
transfected 293T cells were allowed to bind radiolabeled
oligonucleotides containing either one or three BCL6 binding sites.
Different concentrations of WP or MP were added to the reaction and
unlabeled DNA probe, anti-BCL6 antibodies or control IgG were used
to verify the specificity of the observed complexes. Interestingly,
addition of WP caused a dose dependent reduction in the main
BCL6-DNA complex in favor of a faster migrating specific complex,
with no apparent reduction in overall DNA binding. Furthermore, DNA
binding by the BCL6 zinc finger only constructs was unaffected by
WP or MP peptides, nor was there any change in the size of the
DNA-protein complex (not shown). Results with the single or triple
binding site probe were identical, and only the former is shown in
FIG. 9A. The same result was seen in nuclear extracts with
endogenous BCL6 from Ly1 cells (data not shown).
[0190] Disruption of the high molecular weight complex by lateral
groove blockade is likely due to loss of BBD corepressors from the
BCL6 complex. However, attempts to supershift SMRT and N-CoR from
these high molecular weight complexes were unsuccessful (consistent
with the experience of other investigators) with the available
antibodies. Furthermore, from the physiological standpoint the
relevant BCL6 complexes in transcriptional repression are those
that form on the promoters of endogenous BCL6 target genes.
Endogenous complexes were analyzed by chromatin
immunoprecipitations (ChIPs) in the BCL6 positive Ly1 and Ly8 cells
after one-hour exposure to WP, MP or buffer. The cross-linked
chromatin was pulled down with antibodies against BCL6, SMRT, N-CoR
and HA (for the peptide). PCR was performed on the purified DNA
using specific primers for BCL6 binding sites of the MIPla
promoter, which we have used previously to map BCL6 recruited
proteins by ChIPs (FIG. 9B). BCL6 itself bound the MIP1.alpha.
promoter in the presence of both WP and MP peptides, confirming
that lateral groove blockade does not interfere with binding to
target genes. In contrast, SMRT and N-CoR were both excluded from
the MIPla promoter in the presence of WP but not MP peptide.
Finally, the WP but not MP peptide was associated with the
MIP1.alpha. promoter by ChIPs consistent with its binding to the
BTB domain and displacing corepressors. These results indicate that
endogenous BCL6 repressosome formation includes the SMRT and N-CoR
proteins and that this complex is disrupted by lateral groove
blockade.
[0191] Transcriptional repressors recruit corepressors to mediate
changes in chromatin structure that lead to silencing of the
targeted locus. Two such modifications associated with silencing
include deacetylation of histone tails and the methylation of
lysine 9 of the tail of histone 3. Accordingly, the SMRT
corepressor is directly implicate in histone deacetylation. ChIP
assays performed in Ly1 and Ly8 cells on the MIPla promoter
indicate that histone 4 tails are deacetylated at baseline but
become acetylated in the presence of WP peptide, indicating that
BBD corepressors mediate deacetylation of histones by BCL6. We also
found that BCL6 target genes were methylated on lysine 9 of histone
3. Although a connection between SMRT/N-CoR and histone methylation
has not yet been reported, we found that these corepressors were
also required for H3-K9 methylation by BCL6. These results suggest
that these very large BBD corepressors repress transcription of
BCL6 target genes by acting as a scaffold for histone deacetylases
and histone methyltransferases. Loss of this scaffold through
lateral groove blockade leads to erasure of these chromatin
modifications with consequent target gene reactivation.
[0192] BCL6 is the most commonly mutated gene in B-cell lymphoma,
based on which it is widely implicated as a potential oncogene.
This view is supported by the identity of certain BCL6 target genes
involved in cell cycle control, apoptosis and differentiation. We
reasoned that if BCL6 repression is oncogenic, re-activation of
target genes by lateral groove blockade might profoundly alter the
phenotype of lymphoma cells. Tumors most likely to respond to WP
peptides are those derived from patients with activating mutations
in the BCL6 gene, which are presumably BCL6-dependent and include
40% of DLBCL cases, 16% of follicular lymphoma cases, and a
significant percentage of AIDS-related lymphomas. In contrast,
B-cell lymphomas that express BCL6 but do not have mutations, or
that do not express BCL6 might not be BCL6 dependent.
[0193] To determine if this is case, the biological effects of
lateral groove inhibition were tested in the following DLBCL cells:
Ly1 and Ly8 (BCL6 mutated), Ly4 (BCL6 negative), Ly7 (BCL6
positive-not mutated), Ly10 (activated B cell type DLBCL); Germinal
center Burkitt Lymphoma Daudi and Raji Cells (BCL6 positive - not
mutated), Ly12 cells (T-cell lymphoma - low BCL6 expression) and
U937 monocytic leukemia cells. Lymphoma cells were treated for 48
hours with peptide buffer or 1 .mu.M WP or MP peptides and the
effects of these treatments on survival, cell cycle and
differentiation analyzed.
[0194] Apoptosis was measured by sub G1/G0 population scoring and
Annexin V staining. Remarkably, within 48 hours, 40-50% of the Ly1
cells underwent programmed cell death with WP but not MP or PB.
Furthermore, flow cytometry of propidium iodide stained Ly 1 cells
of WP indicated that a minority of cells were still viable (FIG.
10A). In contrast, the predicted non-BCL6 dependent cell lines were
not affected by peptide treatment, with the exception of Raji cells
(20% apoptosis with WP). This result is consistent with Shaffer et
al (2000) where a BCL6 zinc fingers- estrogen receptor activation
domain fusion protein caused mild apoptosis, and may indicate that
BCL6 may participate in maintaining the malignant phenotype in a
subset of tumors where the gene is wild type. Similar results were
obtained in XTT metabolic viability assays (10B). Cell cycle
analysis was performed by propidium iodide staining followed by
flow cytometry (FIG. 10C). WP but not mutant peptide induced G1
arrest in Ly1, Ly8 and Raji cells but not in the other cells. These
results show that BCL6 is necessary to maintain the malignant
phenotype of this kind of B-cell lymphomas. BCL6 is hypothesized to
block differentiation of B-cells by repressing the Blimp-1 master
regulator of plasma cell differentiation. Our flow cytometry
analysis of differentiation markers CD10, CD38 and CD138 indicate
that lateral groove blockade does not induce differentiation in any
of the B-cell lines analyzed (FIG. 10D). This indicates that either
BCL6 is not involved in blocking differentiation, or that putative
BCL6 target genes involved in differentiation are not regulated by
BBD corepressor recruitment. Taken together, these results suggest
that BCL6 expression is required for survival and proliferation of
B-cell lymphomas containing BCL6 activating mutations, and may play
a role in a subset of non-mutated B-cell lymphomas.
[0195] Transcriptional repression occurs through the coordinated
actions of protein complexes recruited to specific target genes by
sequence specific transcription factors. The composition of these
repressosomes may vary from locus to locus, suggesting that
different cofactors are involved in regulating different sets of
target genes. The specific set of protein-protein interactions that
occur in these repressosomes determine the transcriptional outcome
as detected by changes in chromatin structure and mRNA expression.
We designed a peptide reagent, able to specifically and "cleanly"
subtract the contribution of the SMRT/N-CoR/BCoR corepressors from
the transcriptional actions of the BCL6 protein. Our results offer
direct evidence of the role of these corepressors in endogenous
target gene chromatin modification, endogenous transcriptional
regulation, and in transcription factor specific biological
effects. The fact that lateral groove blockade uncovered the
requirement of BCL6 transcriptional repression for survival of
DLBCL cells with BCL6 mutations suggest that BCL6 is truly a
lymphoma oncogene and not just a marker of cells that have
traversed germinal center differentiation.
[0196] Finally, in experimental cancer therapy, much attention has
been focused on therapeutic re-expression of silenced genes
(transcription therapy) with a particular emphasis on the
development of drugs that inhibit enzymatic activities, such as
histone deacetylase inhibitors. However, the most successful
transcription therapy drug is also the only one that directly
targets the interaction of a transcriptional repressor with its
partner corepressors--i.e. all trans retinoic acid targeting of the
PML/RAR.alpha. oncoprotein in acute promyelocytic leukemia.
Similarly, our results suggest that our lateral groove blocking
peptide drug is a novel and promising structure guided specific
transcription therapy agent. Therefore, we have initiated
pre-clinical studies with chemical derivatives with the intent to
move into clinical trials in patients with DLBCL.
[0197] Materials and Methods
[0198] Expression and purification of peptides. The (His)6-pTAT-HA
bacterial expression plasmid were obtained from Dr. Steve Dowdy
(UCSD). Oligonucleotides containing the wild type [0199]
(5'-CATGGCTGGTGGCCACGGTGAAGGAGGCGGGCCGCTCCATCCATGAGATCCCGCGCG
AGGAGCTGCGGCACACGCCCGAGCTGCCCCTGGCCC-3' and
5'-TCGAGGGCCAGGGGCAGCTCGGGCGTGTGCCGCAGCTCCTCGCGCGGGATCTCATGG
ATGGAGCGGCCCGCCTCCTTCACCGTGGCCACCAGC-3') or mutant BBD
(5'-CATGGCTGGTGGCCACGGTGAAGGAGGCGGGCCGCTCCATCC
ATGCAGCTGCAGCTGAGGAGCTGCGGCACACGCCCGAGCTGCCCCTGGCCC-3' and
5'-TCGAGGGCCAGGGGCAGCTCGGGCGTGTGCCGCAGCTCCTCAG
CTGCAGCTGCATGGATGGAGCGGCCCGCCTCCTTCACCGTGGCCACCAGC-3') BBD
sequences were inserted into Nco I and XhoI sites, and the
construct verified by automated sequence (Albert Einstein
Sequencing Facility). Peptide expression was induced with IPTG in
BL21 (D3) E. coli cells (Novagen), which were harvested by
centrifugation, washed with PBS and resuspended in lysis buffer (50
mM Tris-HCl pH8, 150 mM NaCl, 5% Glycerol). Lysozyme (1 mg/ml) was
added and incubated for 30 min at 4.degree. C. followed by
sonication using a Vibra Cell sonicator (Sonic & Material Inc).
The sample was centrifuged at 12000 rpm in a Sorvall RCSB
centrifuge, and resuspended in a peptide buffer (PB=20 mM Phosphate
pH=7.4 10 mM imidazole, 150 mM NaCl, 5% Glycerol, 4 M urea) and
affinity purified by Ni-NTA Hi-Trap column (Pharmacia Biotech)
using an AKTA Purifier 10 (AP Biotech). The purity of peptide
fractions was verified by 15% tricine SDS-PAGE followed by
Coomassie Blue staining (Bio-Rad) or by Western Blot using HA
polyclonal antibodies (Sigma).
[0200] Peptide transduction. Cells were exposed to different
concentrations (100 nM, 250 nM, 500 nM, 1 .mu.M) of WP and MP
peptides for 2 hours and harvested at different time points. The
cells were then washed 4 times with PBS, and whole cell lysates
obtained using a Lysis Buffer=50 mM Hepes, 0.1% NP-40, 50 mM NaCl,
or the pellet was fractionated by resuspending the pellet in a
buffer of 10 mM Hepes-KOH pH=7.9, 10 mM KCl, 0.5 mM DTT, 0.5 mM
PMSF, 10 .mu.M Leucpeptin on ice for 10 minutes. After treatment in
a microcentrifuge at maximum speed (15 min.), the supernatant was
saved as the cytoplasm fraction. The pellet was then resuspended in
a buffer of 10 mM Hepes-KOH pH=7.9, 25% glycerol, 410 mM NaCl, 1.5
mM MgCl.sub.2, 0.2 mM EDTA, 055 mM DTT, 0.5 mM PMSF on ice. After
20 minutes the sample was again centrifuged at maximum speed for 15
minutes. The supernatant was saved as the nuclear extract. The
peptides were visualized by loading the fractions as well the whole
cell lysates in 15% tricine SDS-PAGE followed by Western Blot using
HA polyclonal antibodies (Sigma).
[0201] Coimmunoprecipitations. For in vitro coimmunoprecipitations,
Ly1 and 293 T cell nuclear extracts were obtained as described
above and incubated with the PB, or WP and MP peptides at a final
concentration of 0.12 mg/ml. For in vivo coimmunoprecipitations,
293 T cells were transfected with 4 .mu.g of BCL6 expression vector
(REF) using Superfect (Qiagen). Forty-eight hours later the cells
were exposed to PB, or the WP or MP peptides for 1 hour, washed 4
times with PBS, and lysed in a buffer of 50 mM Hepes, 0.1% NP-40,
50 mM NaCl. In vitro and in vivo samples were precleared with a mix
of Protein G/A agarose beads (Roche, Mannheim, Germany), incubated
overnight with BCL6 D-8 monoclonal antibodies (Santa Cruz) and
pulled down with G/A agarose beads (Roche) for 2 hours. The pellet
was washed 3 times with lysis buffer, resuspended in 2.times.
loading buffer and analyzed by Western blot with HA polyclonal
antibodies (Sigma).
[0202] Immunofluorescence. 1.times.10.sup.6 293T Cells were plated
in 22 mm sterile glass coverslips and placed in 6 well dishes. 24
hours later cells were transfected with 100 ng pEF-BCL6 and/or 200
ng CMX-SMRT or pEF vector using the Superfect reagent (Qiagen).
pBluescript was added to 3000 ng per well. Immunostaining was
performed as previously reported (REF). BCL6, SMRT and peptides
were detected using BCL6 monoclonal antibodies (Dako, Fort Collins,
Colo.), SMRT polyclonal antibodies (Upstate Biotechnologies,
Waltham, Mass.) or HA polyclonal antibodies (Sigma), followed by
donkey anti-mouse and/or anti-rabbit secondary antibodies (Jackson
Immuno Research, West Grove, Pa.) conjugated to Cy2 and Cy3
respectively. Confocal microscopy was performed using a Leica AOBS
Laser Scanning Confocal Microscope (Leica) in the Albert Einstein
Analytical Imaging Facility. Images were captured in each channel
independently in non-overlapping spectra. Each experiment was
repeated at least 3 times in duplicates and multiple fields imaged
and captured.
[0203] Reporter assays. 293T cells were plated in a 12 well dish at
a density of 2.times.10.sup.5 per well or in a 24-well dish at
1.times.10.sup.5 per well (Invitrogen, Carlsbad, Calif.), and
transfected with 100 ng of either (GAL4).sub.5-TK-Luc or
(BCL6).sub.4-TK-Luc and cotransfected with the corresponding
expression vectors indicated in the figure legends, plus a renilla
reporter construct as an internal control, using Superfect (Qiagen,
Valencia, Calif.). After 24 hrs cells were treated with PB, MP or
WP at a final concentration 1 .mu.M for 20 hrs, adding fresh
peptide every 2.5 hrs. Cells were harvested and dual luciferase
assays were performed (Promega, Madison, Wis.) according to the
manufacturer's instructions. Luciferase activity was read in a
POLARstar Optima microplate luminometer (BMG Labtechnologies,
Durham N.C.). All transfections were performed in quadruplicate and
repeated at least 3 times. Lysates were subjected to western blot
to verify protein expression from transfected vectors.
[0204] Real-time PCR. 10.sup.7 Ly1, Ly8 or Ly4 cells were harvested
after no treatment or treatment with PB or 1 .mu.M of WP or MP for
7.5 hours, adding fresh peptide every 2.5 hours. RNA was extracted
from the cells using TRIzol.RTM. Reagent (Invitrogen). cDNA was
synthesized from 10 .mu.g of RNA using the Superscript II.TM. First
Strand cDNA Synthesis System kit (Invitrogen). 1 .mu.l of cDNA for
each condition in a final volume of 20 .mu.l was used in real-time
PCR analysis using the QuantiTect SYBR.RTM. Green PCR kit (QIAGEN)
in a DNA Engine Opticon 2.RTM. System thermal cycler (MJ Research).
mRNA levels of BCL6 target genes were normalized to endogenous
levels of HPRT mRNA and calculated relative to buffer using the
.DELTA..DELTA.Ct method. The peptide treatment of the cells was
performed two or three times and real-time PCR experiments were
repeated four times with each reaction conducted in triplicate.
[0205] The following primers were used:
TABLE-US-00002 CD80-F: CATCCTGGGCCATTACCTTA, CD80-R:
TCTCTCTCTGCATCTTGGGG, BCL6-F: GACTCTGAAGAGCCACCTGC, BCL6-R:
CTGGCTTTTGTGACGGAAAT, CYCLIND2-F: CCGGACCTAATCCCTCACTC, CYCLIND2-R:
CACACCGATGCAGCTTTCTA, HPRT-F: AAAGGAACCCCACGAAGTGTT HPRT-R:
TCAAGGGCATATCCTACAACAA
[0206] Chromatin immunoprecipitation. 10.times.10.sup.7 Ly1 and Ly8
cells were treated with PB or 1 .mu.M WP or MP peptides for 7.5
hours adding fresh peptide every 2.5 hours. Cells were fixed in 1%
formaldehyde (Fisher) (50 mM Hepes pH 8.0, 1mM EDTA, 100 mM NaCl,
0.5 mM EGTA, 37% formaldehyde) for 10 min at room temperature and
quenched by 0.125 M glycine for 10 min. Cells were washed twice
with ice-cold PBS and resuspended in 4 ml of lysis buffer (1% SDS,
10 mM EDTA pH 8.0, 50 mM Tris-HC1 pH 8.0) at 4.degree. C. for 10
minutes. The lysates were sonicated 5.times.30 seconds at an
amplitude of 55% in a Ultrasonic Dismembrator Model 500 (Fisher) to
obtain chromatin fragments with an average size of 300-500 bp,
centrifuged at 14,000 rpm for 10 minutes and the supernatants
precleared with a mixture of protein A/G agarose beads (Roche). 10%
input was collected at this point for later analysis. Specific
immunoprecipitations (each on .about.10.times.10.sup.6 cells) were
performed using rabbit polyclonal antibodies for BCL6 (N-3),
.alpha.N-CoR (H-303) (Santa Cruz), SMRTe, histone 4 pan-acetylated
and histone 3 Lysine 9 dimethyl (Upstate Biotechnologies), HA
(Sigma), normal rabbit serum (Jackson Immuno Research) or water
control overnight at 4.degree. C. DNA-protein complexes were
pulled-down by mixing with protein A/G agarose beads at 4.degree.
C. for 30 minutes and washed twice with each of the following
buffers for 10 minutes at RT: 1.-0.1% SDS, 1% Triton X-100, 2 mM
EDTA pH 8.0, 20 mM tris-HCl pH 8.0, 150 mM NaCl; 2.-0.1% SDS, 1%
Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris-HC1 pH 8.0, 500 mM NaCl;
3.-0.25M LiCl, 1% NP-40, 1% Na-Deoxycholate, 1 mM EDTA pH 8,0 and
10 mM tris-HC1 pH 8.0; and finally TE (10 mM Tris, 1 mM EDTA pH
8,0). After the last wash, the beads were resuspended in 100 .mu.l
of Elution Buffer (1% SDS, 0.1M NaHCO.sub.3), incubated overnight
at 65.degree. C. overnight to reverse cross-links and purified
using QIAquick PCR purification columns (Qiagen). The resulting DNA
fragments were detected by 45 cycles of PCR in a GeneAmp 9700
thermal cycler (Perkin-Elmer-ABI). The following primers were used:
MIP-1.alpha. promoter: S-5'-ACGATGCTGGGTCAGGTATC-3'
AS-5'-AGTGACTAGGGCGCTGTGTT-3' (192 by product) and BCL6 exonl:
S-5'-GGGTTCTTAGAAGTGGTGATGC-3' AS-5'-TGGGACTAATCTTCGGCATT-3', and a
BCL6 intron 7 negative control S-5'-CGATGAGGAGTTIVGGGATGT-3'
AS-5'-TTTCTGGGGGCTCTGTGGACT-3'. These experiments were performed
four times and the immunoprecipitations with each antibodies were
performed in duplicates in each of the four experiments.
[0207] Cell viability. Ly1, Raji and U937 cells were treated with
BP, MP, or WP during 48 hours, during that period of time fresh
peptide or BP was added every 3 hours, changing the media every 6
hours. Cell viability was determined using the Tox2 XTT bases kit
(Sigma) following manufactured instruction. Briefly, XTT solution
was added in a final concentration of 20% v/v of the cultured media
cells plated in 96 wells plate. The cells were incubated for 4
hours and the A.sub.450 determined using a MRC Revelation
microplate reader (Dinex Technologies, West Sussex, UK). The
experiment was performed twice in quadruplicate.
[0208] FACS analysis. The cells lines were treated with BP, MP, or
WP for 48 hours, with fresh peptide or BP added every 3 hours, and
the media changed every 6 hours. After treatment, the cells were
harvested, washed with PBS 3% BSA (Sigma), resuspended at
1.times.10.sup.6 cells/50 .mu.L in PBS/3% BSA/0.1% NaN.sub.2. The
cells were immunostained with 5 .mu.l CD10-R-PE and CD38-APC
antibodies (Caltag, Burlingame, Calif.) per 10.sup.6 cells and
incubated on ice for 30 min. The cells were then washed twice with
PBS/3% BSA/0.1% NaN.sub.2 and resuspended in 500 .mu.L PBS/3%
BSA/0.1% NaN.sub.2. The samples were analyzed on a FACSCalibur or
FACScan flow cytometer (Becton Dickinson) using the CellQuest
program (BD Bioscience, San Jose, Calif.).
[0209] For cell cycle analysis and apoptosis quantification the
cells were fixed in cold 70% ethanol and stored until staining.
Before staining, the cells were washed twice in PBS and resuspended
in 1 ml of PBS. The cells were then stained with 50 .mu.L of
propidium iodide (Sigma)(1mg/ml) and 20 .mu.l of ribonuclease
A(Sigma)(10 .mu.g/ml). The cells were incubated for 2 hours at
4.degree. C. and then measured by flow cytometry in a FACScan.
Cells in pre G1/G0 (hypodiploid DNA) were considered apoptotic. The
CellQuest program was used for quantification; cell cycle was
analyzed using ModFit software (Verity Software House, Inc,
Topsham, Me.). All experiments were performed between 3 and 6
times.
EXAMPLE 3
The Interaction of BCL6 with BCoR
[0210] BCoR is a BCL6 co-repressor that was initially identified
from a partial clone that interacted with the BCL6 BTB domain in a
yeast two-hybrid experiment (Huynh et al., 2000). Genes Dev 14,
1810-1823)). BCoR binds to the BTB domain of BCL6, but does not
interact directly with eight other BTB domain proteins that were
tested (Huynh et al., 2000). BCoR can potentiate BCL6 repression,
possibly through interactions with histone deacetylases (HDACs).
Although BCoR has no obvious sequence similarity to the SMRT
corepressor, the interactions of SMRT and BCoR to the BCL6 BTB
domain are mutually exclusive (Huynh et al., 2000), suggesting that
the two corepressors bind to overlapping sites on the BCL6 BTB
domain. BCoR may have additional roles with transcriptional
partners other than BCL6, and is a key transcriptional regulator
during early embryogenesis (Ng et al., 2004).
[0211] The initial BCL6 interacting fragment identified in the
yeast two-hybrid screen consisted of residues 112-753 of BCoR
(Huynh et al., 2000). We expressed subfragments of this region of
BCoR as hexahistidine tagged fusion proteins in E. coli, and using
the co-purification method described for the identification of the
SMRT BBD (Example 1), we found that a purified fusion protein that
contained residues 317 to 547 of BCoR bound directly to the BCL6
BTB domain, while fusion proteins containing residues 112 to 342,
or 542 to 753 of BCoR did not. The BCoR fragment 317-547 did not
bind to the H116A or N21K mutant forms of the BCL6 BTB domain.
Similarly, the SMRT BBD does not bind to these two BCL6 mutants in
a similar assay (Example 1).
[0212] Further mapping of the minimal BCoR fragment was done with a
version of an electrophoretic mobility shift assay (EMSA). Purified
BCL6 BTB domain was mixed with a BCoR fragment (either as a fusion
protein or as a purified peptide) and incubated at room temperature
in a non-denaturing buffer. The mixture was analyzed by
non-denaturing polyacrylamide gel electrophoresis (PAGE) and
protein bands were visualized with either Coomassie blue staining
or silver staining. A change in the position of the BCL6 BTB domain
band was indicative of complex formation. FIG. 11 shows a result
for a binding experiment with a series of thioredoxin-6his-BCoR
fusion proteins. A fragment of BCoR from residues 494-518
(494-CAIYRSEIISTAPSSWVVPGPSPNE-518) forms a complex with the BCL6
BTB domain, while fragments consisting of residues 494-510 or
506-522 do not interact under these conditions. FIG. 12 shows a
titration of increasing amounts of BCoR peptide with a constant
amount of BCL6 BTB domain in an EMSA binding assay. Furthermore,
peptides of sequence RSEIISTAPASAVAPGP or RSEIISTAPWSSVVPGP did not
result in a shift of the BCL6 BTB band in an EMSA assay. These two
sequences correspond to a S507A / W509A / V511A BCoR triple
mutation, and to a S507W / W509S BCoR double mutation,
respectively. This result demonstrates the importance of the region
from 507-SSWVV-511 of BCoR for the interaction with the BCL6 BTB
domain.
[0213] Purified BCL6 BTB domain was cocrystallized with a purified
peptide of sequence RSEIISTAPSSWVVPGP, which corresponds to
residues 498-514 of BCoR. Crystals were obtained with the hanging
drop method by mixing 1 microliter of protein-peptide mixture with
1 microliter of a precipitant solution consisting of sodium
acetate, potassium phosphate and sodium phosphate, and
equilibrating with the precipitant solution. X-ray diffraction data
were collected on crystals at 100 K on a Bruker Proteum CCD
detector . Crystallographic statistics are presented in Table 2.
The crystals form in space group P6(1)22 with unit cell dimensions
of a=b=150.91 .ANG., c=310.34 .ANG.. The structure was solved by
molecular replacement using the BCL6 BTB domain structure. The
asymmetric unit contains four crystallographically unique 2:2 BTB
-peptide complexes, for a total of eight BTB chains and eight BCoR
peptide chains. The structure was refined with CNS and Refmac to a
final R factor of 22.37% and an Rfree of 26.32%. The refined model
has an rms deviation of 0.008 .ANG. on bond lengths, and
1.3.degree. on bond angles. A representative complex from the
crystal structure is shown diagrammatically in FIG. 13.
[0214] There are small but significant differences in the BCL6 BTB
structure in the SMRT and BCoR complexes. The most notable
difference is in the position of the side chain of residue His-116,
which is positioned above the SMRT peptide in the SMRT/BCL6
complex. In contrast, the BCoR peptide covers His116 in the
BCoR/BCL6 complex (FIG. 14). Since binding affinity is to both the
SMRT and BCoR BBDs is severely impaired in the BCL6 H116A mutant,
the H116/corepressor interactions are important in both cases even
though the molecular details are different in each case.
[0215] Many of the interactions between the SMRT and BCoR BBDs with
the BCL6 BTB domain involve main chain residues of the peptides.
This explains in part the very low similarity between the SMRT BBD
and the BCoR BBD. Nevertheless, amino acid substitutions in both
SMRT and BCoR BBDs confirm the importance of key residues in case.
The alignment of the two peptides as based on the superposition of
the crystal structures is shown in FIG. 5. The most important
similarities between the two peptides are at positions His1426 and
pro1429 of SMRT, which are equivalent to Trp509 and Pro 512 of
BCoR, respectively. Note that although Ser1424 of SMRT is nominally
aligned with Ser507 of BCoR, these two residues are in distinct
structural environments. This is due in part to the different
conformations of BCL6 residue His116 in the two complexes as
described above, and in part to significant differences in the path
of the peptide backbone in the two corepressor peptides in this
region.
TABLE-US-00003 TABLE 2 X-ray diffraction statistics. Resolution
3.00 .ANG. Unique reflections 41133 Redundancy 3.5 Completeness
96.3% <|>/<.sigma.|> 13.72 R.sub.sym 6.9%
[0216] FIG. 16 shows is a ligplot (Wallace et al., 1995) of the
interactions of one of the BCoR peptide with a BCL6 BTB dimer
(Chains A and B). There are 8 such equivalent plots, since there
are four independent 2:2 complexes in the crystal asymmetric unit.
This one is representative of the other eight. The FIG. 16
structure does not include any water molecules (vs. the similar
plot at FIG. 4A).
[0217] In view of the above, it will be seen that the several
advantages of the invention are achieved and other advantages
attained.
[0218] As various changes could be made in the above methods and
compositions without departing from the scope of the invention, it
is intended that all matter contained in the above description and
shown in the accompanying drawings shall be interpreted as
illustrative and not in a limiting sense.
[0219] All references cited in this specification are hereby
incorporated by reference. The discussion of the references herein
is intended merely to summarize the assertions made by the authors
and no admission is made that any reference constitutes prior art.
Applicants reserve the right to challenge the accuracy and
pertinence of the cited references.
TABLE-US-00004 APPENDIX SEQ ID NO:s and other relevant sequence
information SEQ ID NO: 1- SMRT corepressor BCL6 binding peptide-
17mer lvatvkeagrsiheipr SEQ ID NO: 2- N-CoR corepressor BCL6
binding peptide- 17mer gittikemgrsiheipr SEQ ID NO: 3- BCoR
corepressor BCL6 binding peptide- 17mer yrseiistapsswvvpg SEQ ID
NO: 4- SMRT corepressor BCL6 binding peptide- 21mer
glvatvkeagrsiheipreel SEQ ID NO: 5- N-CoR corepressor BCL6 binding
peptide- 21mer dgittikemgrsiheiprqdi SEQ ID NO: 6- BCoR corepressor
BCL6 binding peptide- 21mer iyrseiistapsswvvpgpsp SEQ ID NO: 7-
SMRT corepressor BCL6 binding peptide- 29mer
glvatvkeagrsiheipreelrhtpelpl SEQ ID NO: 8- N-CoR corepressor BCL6
binding peptide- 29mer dgittikemgrsiheiprqdiltqesrkt SEQ ID NO: 9-
BCoR corepressor BCL6 binding peptide- 29mer
iyrseiistapsswvvpgpspneenngk SEQ ID NO: 10- peptide consensus
sequence
(l/g/y)(v/i/r)(a/t/s)(t/e)(v/i)(k/i)(e/s)(a/m/t)(g/a)(r/p)s(i/s)(h/w)(e/v)-
(i/v)p(r/g) SEQ ID NO: 11- wt BCL6 BTB domain 10 20 30 40 50 60
MASPADSCIQ FTRHASDVLL NLNRLRSRDI LTDVVIVVSR EQFRAHKTVL MACSGLFYSI
70 80 90 100 110 120 FTDQLKCNLS VINLDPEINP EGFCILLDFM YTSRLNLREG
NIMAVMATAM YLQMEHVVDT 129 CRKFIKASE SEQ ID NO: 12- mutant BCL6 BTB
domain that can be expressed in a soluble form, e.g., in E. coli.
The mutations are C8Q, C67R and C84N). The extra "GS" residues at
the N terminus are non-natural residues introduced by cloning
strategy. GSADSQIQFT RHASDVLLNL NRLRSRDILT DVVIVVSREQ FRAHKTVLMA
CSGLFYSIFT DQLKRNLSVI NLDPEINPEG FNILLDFMYT SRLNLREGNI MAVMATAMYL
QMEHVVDTCR KFIKASE Alignment of wt and mutated BCL6 BTB domain (wt
on top). Three wt Cys were changed by site-directed mutagenesis:
C8Q, C67R and C84N.
MASPADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQ
GSADSQIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQ
LKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKF
LKRNLSVINLDPEINPEGFNILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKF IKASE
IKASE
Sequence CWU 1
1
34117PRTHomo sapiens 1Leu Val Ala Thr Val Lys Glu Ala Gly Arg Ser
Ile His Glu Ile Pro1 5 10 15Arg217PRTHomo sapiens 2Gly Ile Thr Thr
Ile Lys Glu Met Gly Arg Ser Ile His Glu Ile Pro1 5 10
15Arg317PRTHomo sapiens 3Tyr Arg Ser Glu Ile Ile Ser Thr Ala Pro
Ser Ser Trp Val Val Pro1 5 10 15Gly421PRTHomo sapiens 4Gly Leu Val
Ala Thr Val Lys Glu Ala Gly Arg Ser Ile His Glu Ile1 5 10 15Pro Arg
Glu Glu Leu 20521PRTHomo sapiens 5Asp Gly Ile Thr Thr Ile Lys Glu
Met Gly Arg Ser Ile His Glu Ile1 5 10 15Pro Arg Gln Asp Ile
20621PRTHomo sapiens 6Ile Tyr Arg Ser Glu Ile Ile Ser Thr Ala Pro
Ser Ser Trp Val Val1 5 10 15Pro Gly Pro Ser Pro 20729PRTHomo
sapiens 7Gly Leu Val Ala Thr Val Lys Glu Ala Gly Arg Ser Ile His
Glu Ile1 5 10 15Pro Arg Glu Glu Leu Arg His Thr Pro Glu Leu Pro Leu
20 25829PRTHomo sapiens 8Asp Gly Ile Thr Thr Ile Lys Glu Met Gly
Arg Ser Ile His Glu Ile1 5 10 15Pro Arg Gln Asp Ile Leu Thr Gln Glu
Ser Arg Lys Thr 20 25928PRTHomo sapiens 9Ile Tyr Arg Ser Glu Ile
Ile Ser Thr Ala Pro Ser Ser Trp Val Val1 5 10 15Pro Gly Pro Ser Pro
Asn Glu Glu Asn Asn Gly Lys 20 251017PRTArtificialconsensus
sequence 10Leu Val Ala Thr Val Lys Glu Ala Gly Arg Ser Ile His Glu
Ile Pro1 5 10 15Arg11129PRTHomo sapiens 11Met Ala Ser Pro Ala Asp
Ser Cys Ile Gln Phe Thr Arg His Ala Ser1 5 10 15Asp Val Leu Leu Asn
Leu Asn Arg Leu Arg Ser Arg Asp Ile Leu Thr 20 25 30Asp Val Val Ile
Val Val Ser Arg Glu Gln Phe Arg Ala His Lys Thr 35 40 45Val Leu Met
Ala Cys Ser Gly Leu Phe Tyr Ser Ile Phe Thr Asp Gln 50 55 60Leu Lys
Cys Asn Leu Ser Val Ile Asn Leu Asp Pro Glu Ile Asn Pro65 70 75
80Glu Gly Phe Cys Ile Leu Leu Asp Phe Met Tyr Thr Ser Arg Leu Asn
85 90 95Leu Arg Glu Gly Asn Ile Met Ala Val Met Ala Thr Ala Met Tyr
Leu 100 105 110Gln Met Glu His Val Val Asp Thr Cys Arg Lys Phe Ile
Lys Ala Ser 115 120 125Glu12127PRTArtificialsynthetic mutant 12Gly
Ser Ala Asp Ser Gln Ile Gln Phe Thr Arg His Ala Ser Asp Val1 5 10
15Leu Leu Asn Leu Asn Arg Leu Arg Ser Arg Asp Ile Leu Thr Asp Val
20 25 30Val Ile Val Val Ser Arg Glu Gln Phe Arg Ala His Lys Thr Val
Leu 35 40 45Met Ala Cys Ser Gly Leu Phe Tyr Ser Ile Phe Thr Asp Gln
Leu Lys 50 55 60Arg Asn Leu Ser Val Ile Asn Leu Asp Pro Glu Ile Asn
Pro Glu Gly65 70 75 80Phe Asn Ile Leu Leu Asp Phe Met Tyr Thr Ser
Arg Leu Asn Leu Arg 85 90 95Glu Gly Asn Ile Met Ala Val Met Ala Thr
Ala Met Tyr Leu Gln Met 100 105 110Glu His Val Val Asp Thr Cys Arg
Lys Phe Ile Lys Ala Ser Glu 115 120
1251393DNAArtificialoligonucleotide for plasmid construction
13catggctggt ggccacggtg aaggaggcgg gccgctccat ccatgagatc ccgcgcgagg
60agctgcggca cacgcccgag ctgcccctgg ccc
931493DNAArtificialoligonucleotide for plasmid construction
14tcgagggcca ggggcagctc gggcgtgtgc cgcagctcct cgcgcgggat ctcatggatg
60gagcggcccg cctccttcac cgtggccacc agc
931593DNAArtificialoligonucleotide for plasmid construction
15catggctggt ggccacggtg aaggaggcgg gccgctccat ccatgcagct gcagctgagg
60agctgcggca cacgcccgag ctgcccctgg ccc
931693DNAArtificialoligonucleotide for plasmid construction
16tcgagggcca ggggcagctc gggcgtgtgc cgcagctcct cagctgcagc tgcatggatg
60gagcggcccg cctccttcac cgtggccacc agc 931720DNAArtificialprimer
17catcctgggc cattacctta 201820DNAArtificialprimer 18tctctctctg
catcttgggg 201920DNAArtificialprimer 19gactctgaag agccacctgc
202020DNAArtificialprimer 20ctggcttttg tgacggaaat
202120DNAArtificialprimer 21ccggacctaa tccctcactc
202220DNAArtificialprimer 22cacaccgatg cagctttcta
202321DNAArtificialprimer 23aaaggaaccc cacgaagtgt t
212422DNAArtificialprimer 24tcaagggcat atcctacaac aa
222520DNAArtificialprimer 25acgatgctgg gtcaggtatc
202620DNAArtificialprimer 26agtgactagg gcgctgtgtt
202722DNAArtificialprimer 27gggttcttag aagtggtgat gc
222820DNAArtificialprimer 28tgggactaat cttcggcatt
202921DNAArtificialprimer 29cgatgaggag tttcgggatg t
213021DNAArtificialprimer 30tttctggggg ctctgtggac t 213125PRTHomo
sapiens 31Cys Ala Ile Tyr Arg Ser Glu Ile Ile Ser Thr Ala Pro Ser
Ser Trp1 5 10 15Val Val Pro Gly Pro Ser Pro Asn Glu 20
253217PRTHomo sapiens 32Arg Ser Glu Ile Ile Ser Thr Ala Pro Ala Ser
Ala Val Ala Pro Gly1 5 10 15Pro3317PRTHomo sapiens 33Arg Ser Glu
Ile Ile Ser Thr Ala Pro Trp Ser Ser Val Val Pro Gly1 5 10
15Pro3417PRTHomo sapiens 34Arg Ser Glu Ile Ile Ser Thr Ala Pro Ser
Ser Trp Val Val Pro Gly1 5 10 15Pro
* * * * *
References